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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. 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

  3. 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.

  4. Therapeutic Treatment of Arthritic Mice with 15-Deoxy Δ(12,14)-Prostaglandin J2 (15d-PGJ2) Ameliorates Disease through the Suppression of Th17 Cells and the Induction of CD4(+)CD25(-)FOXP3(+) Cells.

    PubMed

    Carregaro, Vanessa; Napimoga, Marcelo H; Peres, Raphael S; Benevides, Luciana; Sacramento, Laís Amorim; Pinto, Larissa G; Grespan, Renata; Cunha, Thiago M; da Silva, João Santana; Cunha, Fernando Q

    2016-01-01

    The prostaglandin, 15-deoxy Δ(12,14)-prostaglandin J2 (15d-PGJ2), is a lipid mediator that plays an important role in the control of chronic inflammatory disease. However, the role of prostanoid in rheumatoid arthritis (RA) is not well determined. We demonstrated the therapeutic effect of 15d-PGJ2 in an experimental model of arthritis. Daily administration of 15d-PGJ2 attenuated the severity of CIA, reducing the clinical score, pain, and edema. 15d-PGJ2 treatment was associated with a marked reduction in joint levels of proinflammatory cytokines. Although the mRNA expression of ROR-γt was profoundly reduced, FOXP3 was enhanced in draining lymph node cells from 15d-PGJ2-treated arthritic mice. The specific and polyclonal CD4(+) Th17 cell responses were limited during the addition of prostaglandin to cell culture. Moreover, in vitro 15d-PGJ2 increased the expression of FOXP3, GITR, and CTLA-4 in the CD4(+)CD25(-) population, suggesting the induction of Tregs on conventional T cells. Prostanoid addition to CD4(+)CD25(-) cells selectively suppressed Th17 differentiation and promoted the enhancement of FOXP3 under polarization conditions. Thus, 15d-PGJ2 ameliorated symptoms of collagen-induced arthritis by regulating Th17 differentiation, concomitant with the induction of Tregs, and, consequently, protected mice from diseases aggravation. Altogether, these results indicate that 15d-PGJ2 may represent a potential therapeutic strategy in RA.

  5. 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

  6. 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

  7. Therapeutic Treatment of Arthritic Mice with 15-Deoxy Δ12,14-Prostaglandin J2 (15d-PGJ2) Ameliorates Disease through the Suppression of Th17 Cells and the Induction of CD4+CD25−FOXP3+ Cells

    PubMed Central

    Benevides, Luciana; Pinto, Larissa G.; Cunha, Thiago M.

    2016-01-01

    The prostaglandin, 15-deoxy Δ12,14-prostaglandin J2 (15d-PGJ2), is a lipid mediator that plays an important role in the control of chronic inflammatory disease. However, the role of prostanoid in rheumatoid arthritis (RA) is not well determined. We demonstrated the therapeutic effect of 15d-PGJ2 in an experimental model of arthritis. Daily administration of 15d-PGJ2 attenuated the severity of CIA, reducing the clinical score, pain, and edema. 15d-PGJ2 treatment was associated with a marked reduction in joint levels of proinflammatory cytokines. Although the mRNA expression of ROR-γt was profoundly reduced, FOXP3 was enhanced in draining lymph node cells from 15d-PGJ2-treated arthritic mice. The specific and polyclonal CD4+ Th17 cell responses were limited during the addition of prostaglandin to cell culture. Moreover, in vitro 15d-PGJ2 increased the expression of FOXP3, GITR, and CTLA-4 in the CD4+CD25− population, suggesting the induction of Tregs on conventional T cells. Prostanoid addition to CD4+CD25− cells selectively suppressed Th17 differentiation and promoted the enhancement of FOXP3 under polarization conditions. Thus, 15d-PGJ2 ameliorated symptoms of collagen-induced arthritis by regulating Th17 differentiation, concomitant with the induction of Tregs, and, consequently, protected mice from diseases aggravation. Altogether, these results indicate that 15d-PGJ2 may represent a potential therapeutic strategy in RA. PMID:27872515

  8. 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.

  9. 15d-PGJ2 Reduced Microglia Activation and Alleviated Neurological Deficit of Ischemic Reperfusion in Diabetic Rat Model

    PubMed Central

    Huang, Lihong; Li, Gang; Feng, Xiaofang; Wang, Luojun

    2015-01-01

    To investigate the effect of PPARγ agonist 15d-PGJ2 treatment on the microglia activation and neurological deficit of ischemia reperfusion in diabetic rat model, adult Sprague-Dawley rats were sacrificed for the research. The rats were randomly categorized into four groups: (1) sham-operated group; (2) standard ischemia group; (3) diabetic ischemia group; (4) diabetic ischemia group with diabetes and treated with 15d-PGJ2. Compared to the sham-operated group, all the ischemic groups have significantly severer neurological deficits, more TNF-α and IL-1 expression, increased labeling of apoptotic cells, increased CD68 positive staining of brain lesion, and increased volume of infarct and cerebral edema in both 24 hours and 7 days after reperfusion. Interestingly, reduced neurological deficits, decreased TNF-α and IL-1 expression, less apoptotic cells and CD68 positive staining, and alleviated infarct and cerebral edema volume were observed when 15d-PGJ2 was intraperitoneally injected after reperfusion in diabetic ischemia group, suggesting its neuroprotective role in regulating microglia activation, which may have a therapeutic application in the future. PMID:26844229

  10. Oxidative stress-induced expression of HSP70 contributes to the inhibitory effect of 15d-PGJ2 on inducible prostaglandin pathway in chondrocytes.

    PubMed

    Bianchi, A; Moulin, D; Hupont, S; Koufany, M; Netter, P; Reboul, P; Jouzeau, J-Y

    2014-11-01

    The inhibitory effect of 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) on proinflammatory gene expression has been extensively documented and frequently ascribed to its ability to prevent NF-κB pathway activation. We and others have previously demonstrated that it was frequently independent of the peroxisome proliferator activated receptor (PPAR)γ activation. Here, we provide evidence that induction of intracellular heat shock protein (HSP)70 by oxidative stress is an additional regulatory loop supporting the anti-inflammatory effect of 15d-PGJ2 in chondrocytes. Using real-time quantitative PCR and Western blotting, we showed that 15d-PGJ2 stimulated HSP70, but not HSP27 expression while increasing oxidative stress as measured by spectrofluorimetry and confocal spectral imaging. Using N-acetylcysteine (NAC) as an antioxidant, we demonstrated further that oxidative stress was thoroughly responsible for the increased expression of HSP70. Finally, using an HSP70 antisense strategy, we showed that the inhibitory effect of 15d-PGJ2 on IL-1-induced activation of the NF-κB pathway, COX-2 and mPGES-1 expression, and PGE2 synthesis was partly supported by HSP70. These data provide a new anti-inflammatory mechanism to support the PPARγ-independent effect of 15d-PGJ2 in chondrocyte and suggest a possible feedback regulatory loop between oxidative stress and inflammation via intracellular HSP70 up-regulation. This cross talk is consistent with 15d-PGJ2 as a putative negative regulator of the inflammatory reaction. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Study of new interactions of glitazone's stereoisomers and the endogenous ligand 15d-PGJ2 on six different PPAR gamma proteins.

    PubMed

    Álvarez-Almazán, Samuel; Bello, Martiniano; Tamay-Cach, Feliciano; Martínez-Archundia, Marlet; Alemán-González-Duhart, Diana; Correa-Basurto, José; Mendieta-Wejebe, Jessica Elena

    2017-10-15

    Diabetes mellitus is a chronic disease characterized by hyperglycemia, insulin resistance and hyperlipidemia. Glitazones or thiazolidinediones (TZD) are drugs that act as insulin-sensitizing agents whose molecular target is the peroxisome proliferator-activated receptor gamma (PPARγ). The euglycemic action of TZD has been linked with the induction of type 4 glucose transporter. However, it has been shown that the effect of TZD depends on the specific stereoisomer that interacts with PPARγ. Therefore, this work is focused on exploring the interactions and geometry adopted by glitazone's stereoisomers and one endogenous ligand on different conformations of the six crystals of the PPARγ protein using molecular docking and molecular dynamics (MD) simulations accompanied by the MMGBSA approach. Specifically, the 2,4-thiazolidinedione ring, pioglitazone (PIO), rosiglitazone (ROSI) and troglitazone (TRO) stereoisomers (exogenous ligands), as well as the endogenous ligand 15d-PGJ2, were evaluated. The six crystallographic structures of PPARγ are available at Protein Data Bank as the PDB entries 2PRG, 4PRG, 3T03, 1I7I, 1FM6, and 4EMA. According to the results, a boomerang shape and a particular location of ligands were found with low variations according to the protein conformations. The 15d-PGJ2, TZD, PIO, ROSI and (S,S)-TRO enantiomers were mostly stabilized by twenty hydrophobic residues: Phe226, Pro227, Leu228, Ile281, Phe282, Cys285, Ala292, Ile296, Ile326, Tyr327, Met329, Leu330, Leu333, Met334, Val339, Ile341, Met348, Leu353, Phe363 and Met364. Most hydrogen bond interactions were found between the polar groups of ligands with Arg288, Ser289, Lys367, Gln286, His323, Glu343 and His449 residues. An energetic analysis revealed binding free energy trends that supported known experimental findings of other authors describing better binding properties for PIO, ROSI and (S,S)-TRO than for 15d-PGJ2 and the TZD ring. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. 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.

  13. BEND3 mediates transcriptional repression and heterochromatin organization

    PubMed Central

    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

  14. Very low density lipoprotein receptor promotes adipocyte differentiation and mediates the proadipogenic effect of peroxisome proliferator-activated receptor gamma agonists.

    PubMed

    Tao, Huan; Hajri, Tahar

    2011-12-15

    Very low density lipoprotein receptor (VLDLR) is a member of the low density receptor family, expressed mostly in adipose tissue, heart, and skeletal muscles. VLDLR binds apolipoprotein-E-triglyceride-rich lipoproteins and plays a key role in lipid metabolism. In adipocytes, VLDLR expression increases with differentiation but it is not known whether it plays a role in the adipogenesis. Here we report that VLDLR expression in 3T3-L1 adipocytes is upregulated by PPARγ agonist 15-deoxy-delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) in dose- and time-dependant manners. Knockdown of peroxisome proliferator-activated receptor-γ (PPARγ) with siRNA abolished pioglitazone- and 15d-PGJ(2)-induced VLDLR expression and simultaneously reduced VLDL uptake in adipocytes. In addition, PPARγ-agonist treatment of control mouse adipocytes (vldlr(+/+)) enhanced adipogenesis and VLDL uptake concurrently with the induction of VLDLR expression. However, vldlr deficiency (vldlr(-/-)) significantly blunted the proadipogenic effects of PPARγ agonists. Sequence analysis revealed the presence of a putative PPARγ responsive sequence (PPRE) within the vldlr promoter, which is responsive to natural (15d-PGJ(2)) and synthetic (pioglitazone) PPARγ agonists. Reporter gene assays using serial deletion of the 5'-flanking region showed that this putative PPRE site induced promoter transactivation, while a site-targeted mutation abolished transactivation. Moreover, electrophoresis mobility shift assay (EMSA) and chromatic immunoprecipitation (ChIP) assays showed the specific binding of PPARγ to the PPRE sequence. Together, these results support a crucial function for VLDLR in adipocyte differentiation and mediation of the proadipogenic effect of PPARγ. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Organic acid mediated repression of sugar utilization in rhizobia.

    PubMed

    Iyer, Bhagya; Rajput, Mahendrapal Singh; Jog, Rahul; Joshi, Ekta; Bharwad, Krishna; Rajkumar, Shalini

    2016-11-01

    Rhizobia are a class of symbiotic diazotrophic bacteria which utilize C4 acids in preference to sugars and the sugar utilization is repressed as long as C4 acids are present. This can be manifested as a diauxie when rhizobia are grown in the presence of a sugar and a C4 acid together. Succinate, a C4 acid is known to repress utilization of sugars, sugar alcohols, hydrocarbons, etc by a mechanism termed as Succinate Mediated Catabolite Repression (SMCR). Mechanism of catabolite repression determines the hierarchy of carbon source utilization in bacteria. Though the mechanism of catabolite repression has been well studied in model organisms like E. coli, B. subtilis and Pseudomonas sp., mechanism of SMCR in rhizobia has not been well elucidated. C4 acid uptake is important for effective symbioses while mutation in the sugar transport and utilization genes does not affect symbioses. Deletion of hpr and sma0113 resulted in the partial relief of SMCR of utilization of galactosides like lactose, raffinose and maltose in the presence of succinate. However, no such regulators governing SMCR of glucoside utilization have been identified till date. Though rhizobia can utilize multitude of sugars, high affinity transporters for many sugars are yet to be identified. Identifying high affinity sugar transporters and studying the mechanism of catabolite repression in rhizobia is important to understand the level of regulation of SMCR and the key regulators involved in SMCR.

  16. 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

  17. Activation of PPARγ by endogenous prostaglandin J2 mediates the antileukemic effect of selenium in murine leukemia.

    PubMed

    Finch, Emily R; Tukaramrao, Diwakar B; Goodfield, Laura L; Quickel, Michael D; Paulson, Robert F; Prabhu, K Sandeep

    2017-03-30

    Supplementation with nontoxic doses of micronutrient selenium has been shown to alleviate chronic myelogenous leukemia (CML) via the elimination of leukemia stem cells (LSCs) in mice. This treatment provides a new and novel method for eliminating the LSCs that are otherwise not targeted by existing therapies. The antileukemic effect of selenium was dependent on the production of endogenous cyclopentenone prostaglandins (CyPGs), Δ-12 prostaglandin J2 (Δ(12)-PGJ2), and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). Here, we show that these endogenous CyPGs, produced by mice maintained on selenium-supplemented diets, alleviate the symptoms of CML through their ability to activate the nuclear hormone receptor, peroxisome proliferator activated receptor γ (PPARγ). GW9662, a potent PPARγ antagonist, blocked the antileukemic effect of selenium supplementation by significantly reducing CyPGs. This effect was mediated by an increase in 15-prostaglandin dehydrogenase (15-Pgdh) activity, which oxidizes and inactivates Δ(12)-PGJ2 and 15d-PGJ2 In contrast, treatment with the PPARγ agonist pioglitazone mimicked selenium supplementation. This treatment led to decreased 15-Pgdh activity and increased CyPG levels, which inhibited CML progression. Selenium-dependent activation of PPARγ mediated by endogenous CyPGs decreased Stat5 expression leading to the downregulation of Cited2, a master regulator of LSC quiescence. These studies suggest a potential role for selenium supplementation as an adjuvant therapy in CML. © 2017 by The American Society of Hematology.

  18. PPAR-γ ligands repress TGFβ-induced myofibroblast differentiation by targeting the PI3K/Akt pathway: implications for therapy of fibrosis.

    PubMed

    Kulkarni, Ajit A; Thatcher, Thomas H; Olsen, Keith C; Maggirwar, Sanjay B; Phipps, Richard P; Sime, Patricia J

    2011-01-06

    Transforming growth factor beta (TGFβ) induced differentiation of human lung fibroblasts to myofibroblasts is a key event in the pathogenesis of pulmonary fibrosis. Although the typical TGFβ signaling pathway involves the Smad family of transcription factors, we have previously reported that peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands inhibit TGFβ-mediated differentiation of human lung fibroblasts to myofibroblasts via a Smad-independent pathway. TGFβ also activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway leading to phosphorylation of Akt(S473). Here, we report that PPAR-γ ligands, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and 15-deoxy-(12,14)-15d-prostaglandin J(2) (15d-PGJ(2)), inhibit human myofibroblast differentiation of normal and idiopathic pulmonary fibrotic (IPF) fibroblasts, by blocking Akt phosphorylation at Ser473 by a PPAR-γ-independent mechanism. The PI3K inhibitor LY294002 and a dominant-negative inactive kinase-domain mutant of Akt both inhibited TGFβ-stimulated myofibroblast differentiation, as determined by Western blotting for α-smooth muscle actin and calponin. Prostaglandin A(1) (PGA(1)), a structural analogue of 15d-PGJ(2) with an electrophilic center, also reduced TGFβ-driven phosphorylation of Akt, while CAY10410, another analogue that lacks an electrophilic center, did not; implying that the activity of 15d-PGJ(2) and CDDO is dependent on their electrophilic properties. PPAR-γ ligands inhibited TGFβ-induced Akt phosphorylation via both post-translational and post-transcriptional mechanisms. This inhibition is independent of MAPK-p38 and PTEN but is dependent on TGFβ-induced phosphorylation of FAK, a kinase that acts upstream of Akt. Thus, PPAR-γ ligands inhibit TGFβ signaling by affecting two pro-survival pathways that culminate in myofibroblast differentiation. Further studies of PPAR-γ ligands and small electrophilic molecules may lead to a new generation of anti

  19. The transcription factor DREAM represses A20 and mediates inflammation

    PubMed Central

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

    2014-01-01

    Here we show that the transcription-repressor DREAM binds to the A20 promoter to repress the expression of A20, the deubiquitinase suppressing inflammatory NF-κB signaling. DREAM-deficient (Dream−/−) mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, USF1 binding to the DRE-associated E-box domain activated A20 expression in response to inflammatory stimuli. These studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy in diseases such as acute lung injury associated with unconstrained NF-κB activity. PMID:24487321

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

    PubMed

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

    2016-01-01

    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. 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. 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 myofibroblast phenoconversion can be both

  1. Musashi mediates translational repression of the Drosophila hypoxia inducible factor

    PubMed Central

    Bertolin, Agustina P.; Katz, Maximiliano J.; Yano, Masato; Pozzi, Berta; Acevedo, Julieta M.; Blanco-Obregón, Dalmiro; Gándara, Lautaro; Sorianello, Eleonora; Kanda, Hiroshi; Okano, Hideyuki; Srebrow, Anabella; Wappner, Pablo

    2016-01-01

    Adaptation to hypoxia depends on a conserved α/β heterodimeric transcription factor called Hypoxia Inducible Factor (HIF), whose α-subunit is regulated by oxygen through different concurrent mechanisms. In this study, we have identified the RNA binding protein dMusashi, as a negative regulator of the fly HIF homologue Sima. Genetic interaction assays suggested that dMusashi participates of the HIF pathway, and molecular studies carried out in Drosophila cell cultures showed that dMusashi recognizes a Musashi Binding Element in the 3′ UTR of the HIFα transcript, thereby mediating its translational repression in normoxia. In hypoxic conditions dMusashi is downregulated, lifting HIFα repression and contributing to trigger HIF-dependent gene expression. Analysis performed in mouse brains revealed that murine Msi1 protein physically interacts with HIF-1α transcript, suggesting that the regulation of HIF by Msi might be conserved in mammalian systems. Thus, Musashi is a novel regulator of HIF that inhibits responses to hypoxia specifically when oxygen is available. PMID:27141964

  2. Musashi mediates translational repression of the Drosophila hypoxia inducible factor.

    PubMed

    Bertolin, Agustina P; Katz, Maximiliano J; Yano, Masato; Pozzi, Berta; Acevedo, Julieta M; Blanco-Obregón, Dalmiro; Gándara, Lautaro; Sorianello, Eleonora; Kanda, Hiroshi; Okano, Hideyuki; Srebrow, Anabella; Wappner, Pablo

    2016-09-19

    Adaptation to hypoxia depends on a conserved α/β heterodimeric transcription factor called Hypoxia Inducible Factor (HIF), whose α-subunit is regulated by oxygen through different concurrent mechanisms. In this study, we have identified the RNA binding protein dMusashi, as a negative regulator of the fly HIF homologue Sima. Genetic interaction assays suggested that dMusashi participates of the HIF pathway, and molecular studies carried out in Drosophila cell cultures showed that dMusashi recognizes a Musashi Binding Element in the 3' UTR of the HIFα transcript, thereby mediating its translational repression in normoxia. In hypoxic conditions dMusashi is downregulated, lifting HIFα repression and contributing to trigger HIF-dependent gene expression. Analysis performed in mouse brains revealed that murine Msi1 protein physically interacts with HIF-1α transcript, suggesting that the regulation of HIF by Msi might be conserved in mammalian systems. Thus, Musashi is a novel regulator of HIF that inhibits responses to hypoxia specifically when oxygen is available.

  3. BRCA1-mediated repression of select X chromosome genes

    PubMed Central

    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

    2004-01-01

    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

  4. MarA-mediated transcriptional repression of the rob promoter.

    PubMed

    Schneiders, Thamarai; Levy, Stuart B

    2006-04-14

    The Escherichia coli transcriptional regulator MarA affects functions that include antibiotic resistance, persistence, and survival. MarA functions as an activator or repressor of transcription utilizing similar degenerate DNA sequences (marboxes) with three different binding site configurations with respect to the RNA polymerase-binding sites. We demonstrate that MarA down-regulates rob transcripts both in vivo and in vitro via a MarA-binding site within the rob promoter that is positioned between the -10 and -35 hexamers. As for the hdeA and purA promoters, which are repressed by MarA, the rob marbox is also in the "backward" orientation. Protein-DNA interactions show that SoxS and Rob, like MarA, bind the same marbox in the rob promoter. Electrophoretic mobility shift analyses with a MarA-specific antibody demonstrate that MarA and RNA polymerase form a ternary complex with the rob promoter DNA. Transcription experiments in vitro and potassium permanganate footprinting analysis show that MarA affects the RNA polymerase-mediated closed to open complex formation at the rob promoter.

  5. Multiple mechanisms mediate glucose repression of the yeast GAL1 gene.

    PubMed Central

    Lamphier, M S; Ptashne, M

    1992-01-01

    Several mechanisms contribute to the glucose repression of the GAL1 gene in Saccharomyces cerevisiae. We show that one mechanism involves the transcriptional down-regulation of the GAL4 gene and a second requires the GAL80 gene. We also examine the contribution of cis-acting negative elements in the GAL1 promoter to glucose repression. In an otherwise wild-type strain disruption of any one of these three mechanisms alleviates repression of GAL1 only 2- to 4-fold. However, in the absence of the other two mechanisms the transcriptional down-regulation of GAL4 is sufficient to repress GAL1 expression 40- to 60-fold and the GAL80-dependent mechanism is sufficient to repress GAL1 expression 20- to 30-fold. These first two mechanisms constitute a functionally redundant system of repression and both must be disrupted in order to abolish glucose repression of GAL1. In contrast, negative elements in the GAL1 promoter are effective in repressing GAL1 expression 2- to 4-fold in glucose medium only when at least one of the other two mechanisms of repression is present. Thus, glucose repression of GAL1 is mediated primarily by the first two mechanisms, whereas the third mechanism supplements repression severalfold. PMID:1631075

  6. TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis.

    PubMed

    Szemenyei, Heidi; Hannon, Mike; Long, Jeff A

    2008-03-07

    The transcriptional response to auxin is critical for root and vascular development during Arabidopsis embryogenesis. Auxin induces the degradation of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors, freeing their binding partners, the AUXIN RESPONSE FACTOR (ARF) proteins, which can activate transcription of auxin response genes. We show that TOPLESS (TPL) can physically interact with IAA12/BODENLOS (IAA12/BDL) through an ETHYLENE RESPONSE FACTOR (ERF)-associated amphiphilic repression (EAR) motif. TPL can repress transcription in vivo and is required for IAA12/BDL repressive activity. In addition, tpl-1 can suppress the patterning defects of the bdl-1 mutant. Direct interaction between TPL and ARF5/MONOPTEROS, which is regulated by IAA12/BDL, results in a loss-of-function arf5/mp phenotype. These observations show that TPL is a transcriptional co-repressor and further our understanding of how auxin regulates transcription during plant development.

  7. Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis

    PubMed Central

    Marques Howarth, Michelle; Simpson, David; Ngok, Siu P.; Nieves, Bethsaida; Chen, Ron; Siprashvili, Zurab; Vaka, Dedeepya; Breese, Marcus R.; Crompton, Brian D.; Alexe, Gabriela; Hawkins, Doug S.; Jacobson, Damon; Brunner, Alayne L.; West, Robert; Mora, Jaume; Stegmaier, Kimberly; Khavari, Paul; Sweet-Cordero, E. Alejandro

    2014-01-01

    Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma–associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes. PMID:25401475

  8. Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis.

    PubMed

    Marques Howarth, Michelle; Simpson, David; Ngok, Siu P; Nieves, Bethsaida; Chen, Ron; Siprashvili, Zurab; Vaka, Dedeepya; Breese, Marcus R; Crompton, Brian D; Alexe, Gabriela; Hawkins, Doug S; Jacobson, Damon; Brunner, Alayne L; West, Robert; Mora, Jaume; Stegmaier, Kimberly; Khavari, Paul; Sweet-Cordero, E Alejandro

    2014-12-01

    Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma-associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes.

  9. 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.

  10. Lipin1-Mediated Repression of Adipogenesis by Rutin.

    PubMed

    Han, Yo-Han; Kee, Ji-Ye; Park, Jinbong; Kim, Dae-Seung; Shin, Soyoung; Youn, Dong-Hyun; Kang, JongWook; Jung, Yunu; Lee, Young-Mi; Park, Jin-Han; Kim, Su-Jin; Um, Jae-Young; Hong, Seung-Heon

    2016-01-01

    Rutin, also called rutoside or quercetin-3-O-rutinoside and sophorin, is a glycoside between the flavonol quercetin and the disaccharide rutinose. Although many effects of rutin have been reported in vitro and in vivo, the anti-adipogenic effects of rutin have not been fully reported. The aim of this study was to confirm how rutin regulates adipocyte related factors. In this study, rutin decreased the expressions of adipogenesis-related genes, including peroxisome proliferators, activated receptor [Formula: see text] (PPAR[Formula: see text], CCAAT/enhancer-binding protein [Formula: see text] (C/EBP[Formula: see text], fatty acid synthase, adipocyte fatty acid-binding protein, and lipoprotein lipase in 3T3-L1 cells. Rutin also repressed the expression of lipin1, which is an upstream regulator that controls PPAR[Formula: see text] and C/EBP[Formula: see text]. In addition, when 3T3-L1 was transfected with lipin1 siRNA to block lipin1 function, rutin did not affect the expressions of PPAR[Formula: see text] and C/EBP[Formula: see text]. These results suggest that rutin has an anti-adipogenic effect that acts through the suppression of lipin1, as well as PPAR[Formula: see text] and C/EBP[Formula: see text].

  11. Feedback repression of ornithine decarboxylase synthesis mediated by antizyme.

    PubMed Central

    Mitchell, J L; Choe, C Y; Judd, G G

    1996-01-01

    The induction of antizyme by spermidine and the resulting enhancement of ornithine decarboxylase (ODC) degradation have been well studied; however, little is known about the mechanism whereby elevated spermidine levels decrease synthesis of the polyamine biosynthetic enzyme. To evaluate the relative contribution of inhibited synthesis, as distinct from enhanced degradation of ODC, spermidine levels were manipulated in a variant cell line that overproduces a stable form of ODC. Spermidine did not selectively inhibit ODC synthesis in these variant cells, supporting the concept that spermidine diminishes ODC synthesis in normal cells owing to enhanced degradation of the protein in the presence of elevated antizyme levels. This model was further investigated in vitro by use of rabbit reticulocyte lysate, which catalyses simultaneous ODC mRNA translation and antizyme-stimulated degradation of ODC protein. Antizyme strongly repressed the incorporation of labelled amino acids into normal rat ODC. Unexpectedly it also diminished the apparent translation of ODC mRNA species coding for enzyme forms that are not destabilized by the post-translational addition of antizyme. The effect of antizyme on ODC translation was not observed in wheatgerm extract, in which there is no antizyme-induced degradation. Further, deletion of a short segment of antizyme necessary for the destabilization of ODC (amino acid residues 113-118) resulted in a form that bound ODC but did not diminish its apparent translation. These results suggest that the co-translational addition of antizyme to ODC results in a complex that is different from, and innately less stable than, that formed when antizyme is added post-translationally. PMID:9003359

  12. Transforming Growth Factor β-Mediated Transcriptional Repression of c-myc Is Dependent on Direct Binding of Smad3 to a Novel Repressive Smad Binding Element

    PubMed Central

    Frederick, Joshua P.; Liberati, Nicole T.; Waddell, David S.; Shi, Yigong; Wang, Xiao-Fan

    2004-01-01

    Smad proteins are the most well-characterized intracellular effectors of the transforming growth factor β (TGF-β) signal. The ability of the Smads to act as transcriptional activators via TGF-β-induced recruitment to Smad binding elements (SBE) within the promoters of TGF-β target genes has been firmly established. However, the elucidation of the molecular mechanisms involved in TGF-β-mediated transcriptional repression are only recently being uncovered. The proto-oncogene c-myc is repressed by TGF-β, and this repression is required for the manifestation of the TGF-β cytostatic program in specific cell types. We have shown that Smad3 is required for both TGF-β-induced repression of c-myc and subsequent growth arrest in keratinocytes. The transcriptional repression of c-myc is dependent on direct Smad3 binding to a novel Smad binding site, termed a repressive Smad binding element (RSBE), within the TGF-β inhibitory element (TIE) of the c-myc promoter. The c-myc TIE is a composite element, comprised of an overlapping RSBE and a consensus E2F site, that is capable of binding at least Smad3, Smad4, E2F-4, and p107. The RSBE is distinct from the previously defined SBE and may partially dictate, in conjunction with the promoter context of the overlapping E2F site, whether the Smad3-containing complex actively represses, as opposed to transactivates, the c-myc promoter. PMID:14993291

  13. 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

  14. Molecular mechanism underlying juvenile hormone-mediated repression of precocious larval–adult metamorphosis

    PubMed Central

    Kayukawa, Takumi; Jouraku, Akiya; Ito, Yuka; Shinoda, Tetsuro

    2017-01-01

    Juvenile hormone (JH) represses precocious metamorphosis of larval to pupal and adult transitions in holometabolous insects. The early JH-inducible gene Krüppel homolog 1 (Kr-h1) plays a key role in the repression of metamorphosis as a mediator of JH action. Previous studies demonstrated that Kr-h1 inhibits precocious larval–pupal transition in immature larva via direct transcriptional repression of the pupal specifier Broad-Complex (BR-C). JH was recently reported to repress the adult specifier gene Ecdysone-induced protein 93F (E93); however, its mechanism of action remains unclear. Here, we found that JH suppressed ecdysone-inducible E93 expression in the epidermis of the silkworm Bombyx mori and in a B. mori cell line. Reporter assays in the cell line revealed that the JH-dependent suppression was mediated by Kr-h1. Genome-wide ChIP-seq analysis identified a consensus Kr-h1 binding site (KBS, 14 bp) located in the E93 promoter region, and EMSA confirmed that Kr-h1 directly binds to the KBS. Moreover, we identified a C-terminal conserved domain in Kr-h1 essential for the transcriptional repression of E93. Based on these results, we propose a mechanism in which JH-inducible Kr-h1 directly binds to the KBS site upstream of the E93 locus to repress its transcription in a cell-autonomous manner, thereby preventing larva from bypassing the pupal stage and progressing to precocious adult development. These findings help to elucidate the molecular mechanisms regulating the metamorphic genetic network, including the functional significance of Kr-h1, BR-C, and E93 in holometabolous insect metamorphosis. PMID:28096379

  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. 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

  17. 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.

  18. Estrogen-mediated epigenetic repression of large chromosomal regions through DNA looping

    PubMed Central

    Hsu, Pei-Yin; Hsu, Hang-Kai; Singer, Gregory A.C.; Yan, Pearlly S.; Rodriguez, Benjamin A.T.; Liu, Joseph C.; Weng, Yu-I; Deatherage, Daniel E.; Chen, Zhong; Pereira, Julia S.; Lopez, Ricardo; Russo, Jose; Wang, Qianben; Lamartiniere, Coral A.; Nephew, Kenneth P.; Huang, Tim H.-M.

    2010-01-01

    The current concept of epigenetic repression is based on one repressor unit corresponding to one silent gene. This notion, however, cannot adequately explain concurrent silencing of multiple loci observed in large chromosome regions. The long-range epigenetic silencing (LRES) can be a frequent occurrence throughout the human genome. To comprehensively characterize the influence of estrogen signaling on LRES, we analyzed transcriptome, methylome, and estrogen receptor alpha (ESR1)-binding datasets from normal breast epithelia and breast cancer cells. This “omics” approach uncovered 11 large repressive zones (range, 0.35∼5.98 megabases), including a 14-gene cluster located on 16p11.2. In normal cells, estrogen signaling induced transient formation of multiple DNA loops in the 16p11.2 region by bringing 14 distant loci to focal ESR1-docking sites for coordinate repression. However, the plasticity of this free DNA movement was reduced in breast cancer cells. Together with the acquisition of DNA methylation and repressive chromatin modifications at the 16p11.2 loci, an inflexible DNA scaffold may be a novel determinant used by breast cancer cells to reinforce estrogen-mediated repression. PMID:20442245

  19. TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1.

    PubMed

    Oh, Eunkyoo; Zhu, Jia-Ying; Ryu, Hojin; Hwang, Ildoo; Wang, Zhi-Yong

    2014-06-18

    Brassinosteroid (BR) regulates plant development by activating the transcription factor brassinazole resistant 1 (BZR1), which activates and represses different target genes to switch cellular programmes. The mechanisms that determine BZR1's transcriptional activities remain largely unknown. Here we show that BZR1 represses target genes by recruiting the Groucho/TUP1-like transcriptional corepressor TOPLESS (TPL). Specific deletion or mutation of an evolutionarily conserved ERF-associated amphiphilic repression (EAR) motif at the carboxy terminus abolishes BZR1's abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1. The EAR motif in BZR1 mediates recruitment of TPL to BZR1-repressed promoters. A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype. BR repression of gene expression also requires histone deacetylases that interact with TPL. Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.

  20. NF-kappaB mediated transcriptional repression of acid modifying hormone gastrin.

    PubMed

    Datta De, Dipanjana; Datta, Arindam; Bhattacharjya, Sumana; Roychoudhury, Susanta

    2013-01-01

    Helicobacter pylori is a major pathogen associated with the development of gastroduodenal diseases. It has been reported that H. pylori induced pro-inflammatory cytokine IL1B is one of the various modulators of acid secretion in the gut. Earlier we reported that IL1B-activated NFkB down-regulates gastrin, the major hormonal regulator of acid secretion. In this study, the probable pathway by which IL1B induces NFkB and affects gastrin expression has been elucidated. IL1B-treated AGS cells showed nine-fold activation of MyD88 followed by phosphorylation of TAK1 within 15 min of IL1B treatment. Furthermore, it was observed that activated TAK1 significantly up-regulates the NFkB subunits p50 and p65. Ectopic expression of NFkB p65 in AGS cells resulted in about nine-fold transcriptional repression of gastrin both in the presence and absence of IL1B. The S536A mutant of NFkB p65 is significantly less effective in repressing gastrin. These observations show that a functional NFkB p65 is important for IL1B-mediated repression of gastrin. ChIP assays revealed the presence of HDAC1 and NFkB p65 along with NCoR on the gastrin promoter. Thus, the study provides mechanistic insight into the IL1B-mediated gastrin repression via NFkB.

  1. NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

    PubMed Central

    Bhattacharjya, Sumana; Roychoudhury, Susanta

    2013-01-01

    Helicobacter pylori is a major pathogen associated with the development of gastroduodenal diseases. It has been reported that H. pylori induced pro-inflammatory cytokine IL1B is one of the various modulators of acid secretion in the gut. Earlier we reported that IL1B-activated NFkB down-regulates gastrin, the major hormonal regulator of acid secretion. In this study, the probable pathway by which IL1B induces NFkB and affects gastrin expression has been elucidated. IL1B-treated AGS cells showed nine-fold activation of MyD88 followed by phosphorylation of TAK1 within 15 min of IL1B treatment. Furthermore, it was observed that activated TAK1 significantly up-regulates the NFkB subunits p50 and p65. Ectopic expression of NFkB p65 in AGS cells resulted in about nine-fold transcriptional repression of gastrin both in the presence and absence of IL1B. The S536A mutant of NFkB p65 is significantly less effective in repressing gastrin. These observations show that a functional NFkB p65 is important for IL1B-mediated repression of gastrin. ChIP assays revealed the presence of HDAC1 and NFkB p65 along with NCoR on the gastrin promoter. Thus, the study provides mechanistic insight into the IL1B-mediated gastrin repression via NFkB. PMID:24009751

  2. SUMO Modification Enhances p66-Mediated Transcriptional Repression of the Mi-2/NuRD Complex

    PubMed Central

    Gong, Zihua; Brackertz, Marc; Renkawitz, Rainer

    2006-01-01

    Human p66α and p66β are two potent transcriptional repressors that interact with the methyl-CpG-binding domain proteins MBD2 and MBD3. An analysis of the molecular mechanisms mediating repression resulted in the identification of two major repression domains in p66α and one in p66β. Both p66α and p66β are SUMO-modified in vivo: p66α at two sites (Lys-30 and Lys-487) and p66β at one site (Lys-33). Expression of SUMO1 enhanced the transcriptional repression activity of Gal-p66α and Gal-p66β. Mutation of the SUMO modification sites or using a SUMO1 mutant or a dominant negative Ubc9 ligase resulted in a significant decrease of the transcriptional repression of p66α and p66β. The Mi-2/NuRD components MBD3, RbAp46, RbAp48, and HDAC1 were found to bind to both p66α and p66β in vivo. Most of the interactions were not affected by the SUMO site mutations in p66α or p66β, with two exceptions. HDAC1 binding to p66α was lost in the case of a p66αK30R mutant, and RbAp46 binding was reduced in the case of a p66βK33R mutant. These results suggest that interactions within the Mi-2/NuRD complex as well as optimal repression are mediated by SUMOylation. PMID:16738318

  3. Glucocorticoid receptor-mediated cis-repression of osteogenic genes requires BRM-SWI/SNF.

    PubMed

    Pico, Michael J; Hashemi, Sharareh; Xu, Fuhua; Nguyen, Kevin Hong; Donnelly, Robert; Moran, Elizabeth; Flowers, Stephen

    2016-12-01

    Glucocorticoids are an effective therapy for a variety of severe inflammatory and autoimmune disorders; however, the therapeutic use of glucocorticoids is severely limited by their negative side effects, particularly on osteogenesis. Glucocorticoids regulate transcription by binding to the glucocorticoid receptor (GR), which then binds the promoters of target genes to induce either activation or repression. The gene activation effects of nuclear hormone receptors broadly require the cooperation of the chromatin remodeling complex known as SWI/SNF, which is powered by an ATPase core. The well-studied SWI/SNF ATPase, BRG1, is required for gene activation by a spectrum of nuclear hormone receptors including GR. However, glucocorticoid-induced side effects specifically related to impaired osteogenesis are mostly linked with GR-mediated repression. We have considered whether cis-repression of osteogenic genes by GR may be mediated by a distinct subclass of SWI/SNF powered by the alternative ATPase, BRM. BRM does not have an essential role in mammalian development, but plays a repressor role in osteoblast differentiation and favors adipogenic lineage selection over osteoblast commitment, effects that mirror the repressor effects of GR. The studies reported here examine three key GR cis-repression gene targets, and show that GR association with these promoters is sharply reduced in BRM deficient cells. Each of these GR-targeted genes act in a different way. Bglap encodes osteocalcin, which contributes to normal maturation of osteoblasts from committed pre-osteoblasts. The Per3 gene product acts in uncommitted mesenchymal stem cells to influence the osteoblast/adipocyte lineage selection point. Fas ligand, encoded by FasL, is a means by which osteoblasts can modulate bone degradation by osteoclasts. Repression of each of these genes by glucocorticoid favors bone loss. The essential role of BRM in cooperation with GR at each of these control points offers a novel

  4. Biosynthesis of 15-deoxy-Δ12,14-PGJ2 and the ligation of PPARγ

    PubMed Central

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

    2003-01-01

    15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) has been identified as an endogenous ligand for PPARγ, inducing adipogenesis in vitro. Additional roles for this molecule in the propagation and resolution of inflammation, ligation of NF-κB, 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 PPARγ-dependent adipocyte activation and is unaltered in clinical settings in which PPARγ activation has been implicated. PMID:12975479

  5. 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.

  6. Peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-Delta12,14-prostaglandin J2, reduces neutrophil migration via a nitric oxide pathway.

    PubMed

    Napimoga, Marcelo H; Vieira, Silvio M; Dal-Secco, Daniela; Freitas, Andressa; Souto, Fabrício O; Mestriner, Fabiola L; Alves-Filho, José C; Grespan, Renata; Kawai, Toshihisa; Ferreira, Sérgio H; Cunha, Fernando Q

    2008-01-01

    Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma), such as 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ2-mediated activation of PPAR-gamma ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ2 administration decreases leukocyte rolling and adhesion to the inflamed mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS-/- mice were not susceptible to 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ2-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-1 expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ2, whereas 15d-PGJ2 inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ2 suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.

  7. 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

  8. 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

  9. LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes.

    PubMed

    Elayyan, Jinan; Lee, Eun-Jin; Gabay, Odile; Smith, Christopher A; Qiq, Omar; Reich, Eli; Mobasheri, Ali; Henrotin, Yves; Kimber, Susan J; Dvir-Ginzberg, Mona

    2017-04-07

    Reduced SIRT1 activity and levels during osteoarthritis (OA), promotes gradual loss of cartilage. Loss of cartilage matrix is accompanied by an increase in matrix metalloproteinase (MMP) 13, partially because of enhanced LEF1 transcriptional activity. In this study, we assessed the role of SIRT1 in LEF1-mediated MMP13 gene expression in human OA chondrocytes. Results showed that MMP13 protein levels and enzymatic activity decreased significantly during SIRT1 overexpression or activation by resveratrol. Conversely, MMP13 gene expression was reduced in chondrocytes transfected with SIRT1 siRNA or treated with nicotinamide (NAM), a sirtuin inhibitor. Chondrocytes challenged with IL-1β, a cytokine involved in OA pathogenesis, enhanced LEF1 protein levels, and gene expression, resulting in increased MMP13 gene expression; however, overexpression of SIRT1 during IL-1β challenge impeded LEF1 levels and MMP13 gene expression. Previous reports showed that LEF1 binds to the MMP13 promoter and transactivates its expression, but we observed that SIRT1 repressed LEF1 protein and mRNA expression, ultimately reducing LEF1 transcriptional activity, as judged by luciferase assay. Finally, mouse articular cartilage from Sirt1(-/-) presented increased LEF1 and MMP13 protein levels, similar to human OA cartilage. Thus, demonstrating for the first time that SIRT1 represses MMP13 in human OA chondrocytes, which appears to be mediated, at least in part, through repression of the transcription factor LEF1, a known modulator of MMP13 gene expression.-Elayyan, J. Lee, E.-J., Gabay, O., Smith, C. A., Qiq, O., Reich, E., Mobasheri, A., Henrotin, Y., Kimber, S. J., Dvir-Ginzberg, M. LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes.

  10. 15-deoxy-?(12,14)-prostaglandin J2 as a potential TRPV1-dependent atopic dermatitis enhancer.

    PubMed

    Shibata, Takahiro; Takahashi, Katsuhiko; Matsubara, Yui; Takahashi, Nobuaki; Mori, Yasuo; Uchida, Koji

    2014-10-01

    Atopic dermatitis (AD) is a recurrent chronic inflammatory skin condition characterized by a complex pathogenesis, including skin barrier dysfunctions, allergy/immunology, and pruritus.In AD lesions, mast cells migrate into the epidermis, and exert their biological effects by releasing paracrine mediators. TRPV1, a non-selective cation channel widely expressed in skin tissues, has been shown to contribute to the development of diverse dermatoses and pruritus. In the present study, we identified a TRPV1 agonist as a neuritogenic enhancer produced from mast cells and characterized a possible molecular mechanism for the TRPV1-dependent neuritogenesis in AD. Based on the hypothesis that activated mast cells produce a TRPV1-dependent neuritogenic enhancer,we screened a number of inflammatory mediators for the neuritogenic-promoting activityand identified a PGD2 metabolite, 15-deoxy-?(12,14)-prostaglandin J2 (15d-PGJ2), as a potential neuritogenic enhancer derived from mast cells. 15d-PGJ2 significantly enhanced the nerve growth factor (NGF)-induced neuritogenesis in PC12 cells, and its enhancing potency was attributed to the electrophilic center of 15d-PGJ2. 15d-PGJ2 indeed activated TRPV1, leading to a significant increase in the intracellular Ca(2) level. In addition, the treatment of PC12 cells with biotinylated 15d-PGJ2 resulted in the formation of a 15d-PGJ2-TRPV1 adduct, indicating that 15d-PGJ2 directly modified the TRPV1 in the cells. Furthermore, 15d-PGJ2 facilitated the NGF-dependent signal transductions including ERK and JNK pathways in a Ca(2 ?)-dependent manner. These findings suggest that 15d-PGJ2 enhances NGF signaling via TRPV1-dependent Ca(2) influx, thereby acting as a potential neuritogenic enhancer in AD. Copyright © 2014. Published by Elsevier Inc.

  11. 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.

  12. A long noncoding RNA induced by TLRs mediates both activation and repression of immune response genes

    PubMed Central

    Carpenter, Susan; Atianand, Maninjay; Aiello, Daniel; Ricci, Emiliano; Gandhi, Pallavi; Hall, Lisa L.; Byron, Meg; Monks, Brian; Henry-Bezy, Meabh; O’Neill, Luke A.J; Lawrence, Jeanne B.; Moore, Melissa J.; Caffrey, Daniel R.; Fitzgerald, Katherine A.

    2015-01-01

    An inducible program of inflammatory gene expression is central to anti-microbial defenses. Signal-dependent activation of transcription factors, transcriptional co-regulators and chromatin modifying factors collaborate to control this response. Here we identify a long noncoding RNA that acts as a key regulator of this inflammatory response. Germline-encoded receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2 mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad acting regulatory component of the circuit that controls the inflammatory response. PMID:23907535

  13. NFIB-Mediated Repression of the Epigenetic Factor Ezh2 Regulates Cortical Development

    PubMed Central

    Barry, Guy; Harvey, Tracey J.; McLeay, Robert; Smith, Aaron G.; Harris, Lachlan; Mason, Sharon; Stringer, Brett W.; Day, Bryan W.; Wray, Naomi R.; Gronostajski, Richard M.; Bailey, Timothy L.; Boyd, Andrew W.

    2014-01-01

    Epigenetic mechanisms are essential in regulating neural progenitor cell self-renewal, with the chromatin-modifying protein Enhancer of zeste homolog 2 (EZH2) emerging as a central player in promoting progenitor cell self-renewal during cortical development. Despite this, how Ezh2 is itself regulated remains unclear. Here, we demonstrate that the transcription factor nuclear factor IB (NFIB) plays a key role in this process. Nfib−/− mice exhibit an increased number of proliferative ventricular zone cells that express progenitor cell markers and upregulation of EZH2 expression within the neocortex and hippocampus. NFIB binds to the Ezh2 promoter and overexpression of NFIB represses Ezh2 transcription. Finally, key downstream targets of EZH2-mediated epigenetic repression are misregulated in Nfib−/− mice. Collectively, these results suggest that the downregulation of Ezh2 transcription by NFIB is an important component of the process of neural progenitor cell differentiation during cortical development. PMID:24553933

  14. The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation.

    PubMed

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

    2014-03-01

    Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.

  15. A structure-function analysis of transcriptional repression mediated by the WT1, Wilms' tumor suppressor protein.

    PubMed

    Madden, S L; Cook, D M; Rauscher, F J

    1993-07-01

    The chromosome 11p13 Wilms' tumor locus (wt1) encodes a zinc finger-containing transcription factor (WT1). WT1 binds to the consensus sequence (5'-GCGGGGGCG-3') and represses transcription when bound to this site in vivo. The mechanism of repression is not yet defined. To investigate the mechanisms of transcriptional repression and map the domains of WT1 responsible, we constructed hybrid proteins between the yeast GAL4 1-147 DNA binding domain and WT1. Fusion of a 298 amino acid glutamine-proline-rich N-terminal segment of WT1 to the GAL4 DNA binding domain created a potent transcriptional repressor. The use of N- and C-terminal truncations of this segment demonstrated that as few as 96 amino acids were required for active repression by GAL4-WT1 hybrid proteins in NIH3T3 fibroblasts. However, the truncated GAL4-WT1 fusion proteins functioned poorly as repressors in embryonic kidney-derived 293 cells, suggesting cell type-specific requirements for transcriptional repression. Site-directed mutagenesis of the WT1 repression domain revealed that deletion of homopolymeric proline and glycine regions, as well as single amino acid changes, partially inactivated the repression function. Single repressor binding sites placed upstream of the transcription start site conferred WT1-mediated repression to a heterologous promoter, whereas multiple sites resulted in additive (non-synergistic) increases in transcriptional repression. Significant repression of transcription was observed when binding sites were placed 760 base pairs upstream or 1000 base pairs downstream relative to the site of transcription initiation. We conclude that the transcriptional repression function of WT1 is contained in the N-terminal, non-DNA binding domain of the protein and that repression can be functionally transferred to a heterologous DNA binding domain.

  16. JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis.

    PubMed

    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

    2012-02-01

    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.

  17. Acute myeloid leukemia requires Hhex to enable PRC2-mediated epigenetic repression of Cdkn2a

    PubMed Central

    Shields, Benjamin J.; Jackson, Jacob T.; Metcalf, Donald; Shi, Wei; Huang, Qiutong; Garnham, Alexandra L.; Glaser, Stefan P.; Beck, Dominik; Pimanda, John E.; Bogue, Clifford W.; Smyth, Gordon K.; Alexander, Warren S.; McCormack, Matthew P.

    2016-01-01

    Unlike clustered HOX genes, the role of nonclustered homeobox gene family members in hematopoiesis and leukemogenesis has not been extensively studied. Here we found that the hematopoietically expressed homeobox gene Hhex is overexpressed in acute myeloid leukemia (AML) and is essential for the initiation and propagation of MLL-ENL-induced AML but dispensable for normal myelopoiesis, indicating a specific requirement for Hhex for leukemic growth. Loss of Hhex leads to expression of the Cdkn2a-encoded tumor suppressors p16INK4a and p19ARF, which are required for growth arrest and myeloid differentiation following Hhex deletion. Mechanistically, we show that Hhex binds to the Cdkn2a locus and directly interacts with the Polycomb-repressive complex 2 (PRC2) to enable H3K27me3-mediated epigenetic repression. Thus, Hhex is a potential therapeutic target that is specifically required for AML stem cells to repress tumor suppressor pathways and enable continued self-renewal. PMID:26728554

  18. EZH2-mediated repression of Dkk1 promotes hepatic stellate cell activation and hepatic fibrosis.

    PubMed

    Yang, Yang; Chen, Xiao-Xia; Li, Wan-Xia; Wu, Xiao-Qin; Huang, Cheng; Xie, Juan; Zhao, Yu-Xin; Meng, Xiao-Ming; Li, Jun

    2017-03-23

    EZH2, a histone H3 lysine-27-specific methyltransferase, is involved in diverse physiological and pathological processes including cell proliferation and differentiation. However, the role of EZH2 in liver fibrosis is largely unknown. In this study, it was identified that EZH2 promoted Wnt pathway-stimulated fibroblasts in vitro and in vivo by repressing Dkk-1, which is a Wnt pathway antagonist. The expression of EZH2 was increased in CCl4 -induced rat liver and primary HSCs as well as TGF-β1-treated HSC-T6, whereas the expression of Dkk1 was reduced. Silencing of EZH2 prevented TGF-β1-induced proliferation of HSC-T6 cells and the expression of α-SMA. In addition, knockdown of Dkk1 promoted TGF-β1-induced activation of HSCs. Moreover, silencing of EZH2 could restore the repression of Dkk-1 through trimethylation of H3K27me3 in TGF-β1-treated HSC-T6 cells. Interestingly, inhibition of EZH2 had almost no effect on the activation of HSC when Dkk1 was silenced. Collectively, EZH2-mediated repression of Dkk1 promotes the activation of Wnt/β-catenin pathway, which is an essential event for HSC activation.

  19. A link between c-Myc-mediated transcriptional repression and neoplastic transformation.

    PubMed Central

    Lee, L A; Dolde, C; Barrett, J; Wu, C S; Dang, C V

    1996-01-01

    Recent studies indicate that the transcription factor c-Myc contributes to oncogenesis by altering the expression of genes involved in cell proliferation, but its precise function in neoplasia remains ambiguous. The ability of c-Myc to bind the sequence CAC(G/A)TG and transactivate appears to be linked to its transforming activity; however, c-Myc also represses transcription in vitro through a pyrimidine-rich cis element termed the initiator (Inr). In transfection experiments using the adenoviral major late (adML) promoter, which contains two Myc binding sites and an Inr, we determined that c-Myc represses transcription through the initiator in vivo. This activity requires the dimerization domain and amino acids 106 to 143, which are located within the transactivation domain and are necessary for neoplastic transformation. We studied a lymphoma-derived c-Myc substitution mutation at 115-Phe, which is within the region required for transcriptional suppression, and found the mutant more effective than wild-type c-Myc in transforming rodent fibroblasts and in suppressing the adML promoter. Our studies of both loss-of-function and gain-of-function c-Myc mutations suggest a link between c-Myc-mediated neoplastic transformation and transcriptional repression through the Inr. PMID:8601634

  20. A link between c-Myc-mediated transcriptional repression and neoplastic transformation.

    PubMed

    Lee, L A; Dolde, C; Barrett, J; Wu, C S; Dang, C V

    1996-04-01

    Recent studies indicate that the transcription factor c-Myc contributes to oncogenesis by altering the expression of genes involved in cell proliferation, but its precise function in neoplasia remains ambiguous. The ability of c-Myc to bind the sequence CAC(G/A)TG and transactivate appears to be linked to its transforming activity; however, c-Myc also represses transcription in vitro through a pyrimidine-rich cis element termed the initiator (Inr). In transfection experiments using the adenoviral major late (adML) promoter, which contains two Myc binding sites and an Inr, we determined that c-Myc represses transcription through the initiator in vivo. This activity requires the dimerization domain and amino acids 106 to 143, which are located within the transactivation domain and are necessary for neoplastic transformation. We studied a lymphoma-derived c-Myc substitution mutation at 115-Phe, which is within the region required for transcriptional suppression, and found the mutant more effective than wild-type c-Myc in transforming rodent fibroblasts and in suppressing the adML promoter. Our studies of both loss-of-function and gain-of-function c-Myc mutations suggest a link between c-Myc-mediated neoplastic transformation and transcriptional repression through the Inr.

  1. Widespread negative response elements mediate direct repression by agonist-liganded glucocorticoid receptor.

    PubMed

    Surjit, Milan; Ganti, Krishna Priya; Mukherji, Atish; Ye, Tao; Hua, Guoqiang; Metzger, Daniel; Li, Mei; Chambon, Pierre

    2011-04-15

    The glucocorticoid (GC) receptor (GR), when liganded to GC, activates transcription through direct binding to simple (+)GRE DNA binding sequences (DBS). GC-induced direct repression via GR binding to complex "negative" GREs (nGREs) has been reported. However, GR-mediated transrepression was generally ascribed to indirect "tethered" interaction with other DNA-bound factors. We report that GC-induces direct transrepression via the binding of GR to simple DBS (IR nGREs) unrelated to (+)GRE. These DBS act on agonist-liganded GR, promoting the assembly of cis-acting GR-SMRT/NCoR repressing complexes. IR nGREs are present in over 1000 mouse/human ortholog genes, which are repressed by GC in vivo. Thus variations in the levels of a single ligand can coordinately turn genes on or off depending in their response element DBS, allowing an additional level of regulation in GR signaling. This mechanism suits GR signaling remarkably well, given that adrenal secretion of GC fluctuates in a circadian and stress-related fashion.

  2. GATA-1-mediated transcriptional repression yields persistent transcription factor IIB-chromatin complexes.

    PubMed

    Martowicz, Melissa L; Grass, Jeffrey A; Bresnick, Emery H

    2006-12-08

    The hematopoietic GATA factors GATA-1 and GATA-2, which have distinct and overlapping roles to regulate blood cell development, are reciprocally expressed during erythropoiesis. GATA-1 directly represses Gata2 transcription, and reduced GATA-2 synthesis promotes red blood cell development. Gata2 repression involves "GATA switches" in which GATA-1 displaces GATA-2 from Gata2 regulatory regions. We show that extragenic GATA switch sites occupied by GATA-2 associate with as much RNA polymerase II (Pol II) and basal transcription factors as present at the active Gata2 promoters. Pol II bound to GATA switch sites in the active locus was phosphorylated on serine 5 of the carboxyl-terminal domain, indicative of elongation competence. GATA-1-mediated displacement of GATA-2 from GATA switch sites reduced Pol II recruitment to all sites except the far upstream -77-kb region. Surprisingly, TFIIB occupancy persisted at most sites upon repression. These results indicate that GATA-2-bound extragenic regulatory elements recruit Pol II, GATA-1 binding expels Pol II, and despite the persistent TFIIB-chromatin complexes, Pol II recruitment is blocked.

  3. The mechanism underlying Ler-mediated alleviation of gene repression by H-NS.

    PubMed

    Shin, Minsang

    2017-01-29

    Secretion of effector proteins in Enteropathogeneic Escherichia coli (EPEC) and Enterohemorrhagic Escherichia coli (EHEC) is mediated by a specialized type III secretion system, components of which are encoded in the LEE operons 1 to 5. H-NS, a global repressor in E. coli, silences the expression of LEE operons. Ler, a master regulator in LEE operons, shares 24% amnio acid identity and 44% amino acid similarity to H-NS. Interestingly, rather than a gene silencer, its main role has been characterized as an antagonizing protein that relieves H-NS-mediated transcriptional silencing. In the previous study we reported molecular mechanism for the repression of LEE5 promoter in EPEC and EHEC by H-NS as a protein interaction between upstream DNA-bound H-NS and the αCTD of promoter-bound RNA polymerase. The mechanism underlying Ler-mediated alleviation of the genes repression by H-NS is largely unknown. We examined regulatory effect of these proteins on LEE5p activity using various in vitro tools. Our results revealed that binding affinity of Ler to the LEE5p DNA is about 40 folds greater than that of H-NS as determined by surface plasmon resonance. We verified that Ler binding removed H-NS bound to the same stretch of DNA on LEE5 promoter resulting in a derepression. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression.

    PubMed

    Kagale, Sateesh; Rozwadowski, Kevin

    2011-02-01

    Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif-mediated transcriptional repression is emerging as one of the principal mechanisms of plant gene regulation. The EAR motif, defined by the consensus sequence patterns of either LxLxL or DLNxxP, is the most predominant form of transcriptional repression motif so far identified in plants. Additionally, this active repression motif is highly conserved in transcriptional regulators known to function as negative regulators in a broad range of developmental and physiological processes across evolutionarily diverse plant species. Recent discoveries of co-repressors interacting with EAR motifs, such as TOPLESS (TPL) and AtSAP18, have begun to unravel the mechanisms of EAR motif-mediated repression. The demonstration of genetic interaction between mutants of TPL and AtHDA19, co-complex formation between TPL-related 1 (TPR1) and AtHDA19, as well as direct physical interaction between AtSAP18 and AtHDA19 support a model where EAR repressors, via recruitment of chromatin remodeling factors, facilitate epigenetic regulation of gene expression. Here, we discuss the biological significance of EAR-mediated gene regulation in the broader context of plant biology and present literature evidence in support of a model for EAR motif-mediated repression via the recruitment and action of chromatin modifiers. Additionally, we discuss the possible influences of phosphorylation and ubiquitination on the function and turnover of EAR repressors.

  5. 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.

  6. Position-effect variegation and z1 mediated white repression in the In(1)wis system in Drosophila melanogaster.

    PubMed

    Rasmuson-Lestander, A; Larsson, J; Rasmuson, B

    1993-01-01

    We have characterized a new X-chromosomal inversion in Drosophila melanogaster, extending from just distal of white to just proximal of the bb locus. The inversion places the w-isoxanthopterinless (wis) allele close to heterochromatin and under the influence of position-effect variegation (PEV). The wis gene activity is also regulated by chromosome pairing-dependent z1-mediated repression. By changing the environment, using specific second site modifiers, altering the amount of heterochromatin, and disturbing the chromosome pairing, we have been able to separately affect the two regulatory phenomena and analyse their respective impact on the wis regulation. We provide evidence that under normal conditions PEV and z1 mediated white repression are additive. However, at extreme levels of wis repression by PEV, changes in the z1-mediated interactions are not observable. This indicates that PEV is epistatic to z1-mediated regulation of wis. We also show that deficiencies in the short arm of Y act as suppressors of the z1-mediated white repression. This suppression does not influence PEV and is thus not due to the lower amount of heterochromatin. We propose that nonhomologous chromosome pairing between X and Y is important for the synapsis-dependent z1-mediated repression of white transcription activity in this system.

  7. m6A RNA methylation promotes XIST-mediated transcriptional repression

    PubMed Central

    Patil, Deepak P.; Chen, Chun-Kan; Pickering, Brian F.; Chow, Amy; Jackson, Constanza; Guttman, Mitchell; Jaffrey, Samie R.

    2017-01-01

    The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that in human cells XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues—a reversible base modification of unknown function in long non-coding RNAs. We show that m6A formation in XIST, as well as in cellular mRNAs, is mediated by RNA binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m6A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m6A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m6A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression. PMID:27602518

  8. ATM mediates repression of DNA end-degradation in an ATP-dependent manner.

    PubMed

    Rahal, Elias A; Henricksen, Leigh A; Li, Yuling; Turchi, John J; Pawelczak, Katherine S; Dixon, Kathleen

    2008-03-01

    Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM+) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated end-joining.

  9. SAFB1 Mediates Repression of Immune Regulators and Apoptotic Genes in Breast Cancer Cells*

    PubMed Central

    Hammerich-Hille, Stephanie; Kaipparettu, Benny A.; Tsimelzon, Anna; Creighton, Chad J.; Jiang, Shiming; Polo, Jose M.; Melnick, Ari; Meyer, Rene; Oesterreich, Steffi

    2010-01-01

    The scaffold attachment factors SAFB1 and SAFB2 are paralogs, which are involved in cell cycle regulation, apoptosis, differentiation, and stress response. They have been shown to function as estrogen receptor corepressors, and there is evidence for a role in breast tumorigenesis. To identify their endogenous target genes in MCF-7 breast cancer cells, we utilized a combined approach of chromatin immunoprecipitation (ChIP)-on-chip and gene expression array studies. By performing ChIP-on-chip on microarrays containing 24,000 promoters, we identified 541 SAFB1/SAFB2-binding sites in promoters of known genes, with significant enrichment on chromosomes 1 and 6. Gene expression analysis revealed that the majority of target genes were induced in the absence of SAFB1 or SAFB2 and less were repressed. Interestingly, there was no significant overlap between the genes identified by ChIP-on-chip and gene expression array analysis, suggesting regulation through regions outside the proximal promoters. In contrast to SAFB2, which shared most of its target genes with SAFB1, SAFB1 had many unique target genes, most of them involved in the regulation of the immune system. A subsequent analysis of the estrogen treatment group revealed that 12% of estrogen-regulated genes were dependent on SAFB1, with the majority being estrogen-repressed genes. These were primarily genes involved in apoptosis, such as BBC3, NEDD9, and OPG. Thus, this study confirms the primary role of SAFB1/SAFB2 as corepressors and also uncovers a previously unknown role for SAFB1 in the regulation of immune genes and in estrogen-mediated repression of genes. PMID:19901029

  10. The Paf1 complex represses small RNA-mediated epigenetic gene silencing

    PubMed Central

    Flury, Valentin; Stadler, Michael Beda; Batki, Julia; Bühler, Marc

    2015-01-01

    RNA interference (RNAi) refers to the ability of exogenously introduced double-stranded RNA (dsRNA) to silence expression of homologous sequences. Silencing is initiated when the enzyme Dicer processes the dsRNA into small interfering RNAs (siRNAs). Small RNA molecules are incorporated into Argonaute protein-containing effector complexes, which they guide to complementary targets to mediate different types of gene silencing, specifically post-transcriptional gene silencing (PTGS) and chromatin-dependent gene silencing1. Although endogenous small RNAs play critical roles in chromatin-mediated processes across kingdoms, efforts to initiate chromatin modifications in trans by using siRNAs have been inherently difficult to achieve in all eukaryotic cells. Using fission yeast, we show that RNAi-directed heterochromatin formation is negatively controlled by the highly conserved RNA polymerase-associated factor 1 complex (Paf1C). Temporary expression of a synthetic hairpin RNA in Paf1C mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes in trans. This repressed state is propagated across generations by continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our data support a model where Paf1C prevents targeting of nascent transcripts by the siRNA-containing RNA-induced transcriptional silencing (RITS) complex and thereby epigenetic gene silencing, by promoting efficient transcription termination and rapid release of the RNA from the site of transcription. We show that although compromised transcription termination is sufficient to initiate the formation of bi-stable heterochromatin by trans-acting siRNAs, impairment of both transcription termination and nascent transcript release is imperative to confer stability to the repressed state. Our work uncovers a novel mechanism for small RNA- mediated epigenome regulation and highlights fundamental roles for Paf1C and the RNAi machinery in building

  11. The Paf1 complex represses small-RNA-mediated epigenetic gene silencing.

    PubMed

    Kowalik, Katarzyna Maria; Shimada, Yukiko; Flury, Valentin; Stadler, Michael Beda; Batki, Julia; Bühler, Marc

    2015-04-09

    RNA interference (RNAi) refers to the ability of exogenously introduced double-stranded RNA to silence expression of homologous sequences. Silencing is initiated when the enzyme Dicer processes the double-stranded RNA into small interfering RNAs (siRNAs). Small RNA molecules are incorporated into Argonaute-protein-containing effector complexes, which they guide to complementary targets to mediate different types of gene silencing, specifically post-transcriptional gene silencing and chromatin-dependent gene silencing. Although endogenous small RNAs have crucial roles in chromatin-mediated processes across kingdoms, efforts to initiate chromatin modifications in trans by using siRNAs have been inherently difficult to achieve in all eukaryotic cells. Using fission yeast, here we show that RNAi-directed heterochromatin formation is negatively controlled by the highly conserved RNA polymerase-associated factor 1 complex (Paf1C). Temporary expression of a synthetic hairpin RNA in Paf1C mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes in trans. This repressed state is propagated across generations by the continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our data support a model in which Paf1C prevents targeting of nascent transcripts by the siRNA-containing RNA-induced transcriptional silencing complex and thereby epigenetic gene silencing, by promoting efficient transcription termination and rapid release of the RNA from the site of transcription. We show that although compromised transcription termination is sufficient to initiate the formation of bi-stable heterochromatin by trans-acting siRNAs, impairment of both transcription termination and nascent transcript release is imperative to confer stability to the repressed state. Our work uncovers a novel mechanism for small-RNA-mediated epigenome regulation and highlights fundamental roles for Paf1C and the RNAi machinery in building

  12. Polycomb Repressive Complex 2-Mediated Chromatin Repression Guides Effector CD8(+) T Cell Terminal Differentiation and Loss of Multipotency.

    PubMed

    Gray, Simon M; Amezquita, Robert A; Guan, Tianxia; Kleinstein, Steven H; Kaech, Susan M

    2017-04-04

    Understanding immunological memory formation depends on elucidating how multipotent memory precursor (MP) cells maintain developmental plasticity and longevity to provide long-term immunity while other effector cells develop into terminally differentiated effector (TE) cells with limited survival. Profiling active (H3K27ac) and repressed (H3K27me3) chromatin in naive, MP, and TE CD8(+) T cells during viral infection revealed increased H3K27me3 deposition at numerous pro-memory and pro-survival genes in TE relative to MP cells, indicative of fate restriction, but permissive chromatin at both pro-memory and pro-effector genes in MP cells, indicative of multipotency. Polycomb repressive complex 2 deficiency impaired clonal expansion and TE cell differentiation, but minimally impacted CD8(+) memory T cell maturation. Abundant H3K27me3 deposition at pro-memory genes occurred late during TE cell development, probably from diminished transcription factor FOXO1 expression. These results outline a temporal model for loss of memory cell potential through selective epigenetic silencing of pro-memory genes in effector T cells.

  13. Antisense-mediated FLC transcriptional repression requires the P-TEFb transcription elongation factor

    PubMed Central

    Wang, Zhi-Wei; Wu, Zhe; Raitskin, Oleg; Sun, Qianwen; Dean, Caroline

    2014-01-01

    The functional significance of noncoding transcripts is currently a major question in biology. We have been studying the function of a set of antisense transcripts called COOLAIR that encompass the whole transcription unit of the Arabidopsis floral repressor FLOWERING LOCUS C (FLC). Alternative polyadenylation of COOLAIR transcripts correlates with different FLC sense expression states. Suppressor mutagenesis aimed at understanding the importance of this sense–antisense transcriptional circuitry has identified a role for Arabidopsis cyclin-dependent kinase C (CDKC;2) in FLC repression. CDKC;2 functions in an Arabidopsis positive transcription elongation factor b (P-TEFb) complex and influences global RNA polymerase II (Pol II) Ser2 phosphorylation levels. CDKC;2 activity directly promotes COOLAIR transcription but does not affect an FLC transgene missing the COOLAIR promoter. In the endogenous gene context, however, the reduction of COOLAIR transcription by cdkc;2 disrupts a COOLAIR-mediated repression mechanism that increases FLC expression. This disruption then feeds back to indirectly increase COOLAIR expression. This tight interconnection between sense and antisense transcription, together with differential promoter sensitivity to P-TEFb, is central to quantitative regulation of this important floral repressor gene. PMID:24799695

  14. Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes.

    PubMed

    Wong, Piu; Hattangadi, Shilpa M; Cheng, Albert W; Frampton, Garrett M; Young, Richard A; Lodish, Harvey F

    2011-10-20

    It is unclear how epigenetic changes regulate the induction of erythroid-specific genes during terminal erythropoiesis. Here we use global mRNA sequencing (mRNA-seq) and chromatin immunoprecipitation coupled to high-throughput sequencing (CHIP-seq) to investigate the changes that occur in mRNA levels, RNA polymerase II (Pol II) occupancy, and multiple posttranslational histone modifications when erythroid progenitors differentiate into late erythroblasts. Among genes induced during this developmental transition, there was an increase in the occupancy of Pol II, the activation marks H3K4me2, H3K4me3, H3K9Ac, and H4K16Ac, and the elongation methylation mark H3K79me2. In contrast, genes that were repressed during differentiation showed relative decreases in H3K79me2 levels yet had levels of Pol II binding and active histone marks similar to those in erythroid progenitors. We also found that relative changes in histone modification levels, in particular, H3K79me2 and H4K16ac, were most predictive of gene expression patterns. Our results suggest that in terminal erythropoiesis both promoter and elongation-associated marks contribute to the induction of erythroid genes, whereas gene repression is marked by changes in histone modifications mediating Pol II elongation. Our data map the epigenetic landscape of terminal erythropoiesis and suggest that control of transcription elongation regulates gene expression during terminal erythroid differentiation.

  15. Anchoring of Heterochromatin to the Nuclear Lamina Reinforces Dosage Compensation-Mediated Gene Repression

    PubMed Central

    Brouhard, Elizabeth A.; Jiang, Jianhao; Sifuentes, Margarita H.

    2016-01-01

    Higher order chromosome structure and nuclear architecture can have profound effects on gene regulation. We analyzed how compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensation in the nematode C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, we found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. We propose a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression. PMID:27690361

  16. 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

  17. The cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 binds to and activates H-Ras

    PubMed Central

    Oliva, José Luis; Pérez-Sala, Dolores; Castrillo, Antonio; Martínez, Natalia; Cañada, F. Javier; Boscá, Lisardo; Rojas, José M.

    2003-01-01

    The cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) induces cell proliferation and mitogen-activated protein kinase activation. Here, we describe that these effects are mediated by 15d-PGJ2-elicited H-Ras activation. We demonstrate that this pathway is specific for H-Ras through the formation of a covalent adduct of 15d-PGJ2 with Cys-184 of H-Ras, but not with N-Ras or K-Ras. Mutation of C184 inhibited H-Ras modification and activation by 15d-PGJ2, whereas serum-elicited stimulation was not affected. These results describe a mechanism for the activation of the Ras signaling pathway, which results from the chemical modification of H-Ras by formation of a covalent adduct with cyclopentenone prostaglandins. PMID:12684535

  18. Id2-Mediated Inhibition of E2A Represses Memory CD8+ T Cell Differentiation

    PubMed Central

    Masson, Frederick; Minnich, Martina; Olshansky, Moshe; Bilic, Ivan; Mount, Adele M.; Kallies, Axel; Speed, Terence P.; Busslinger, Meinrad; Nutt, Stephen L.

    2013-01-01

    The transcription factor inhibitor of DNA binding (Id)2 modulates T cell fate decisions, but the molecular mechanism underpinning this regulation is unclear. In this study we show that loss of Id2 cripples effector differentiation and instead programs CD8+ T cells to adopt a memory fate with increased Eomesodermin and Tcf7 expression. We demonstrate that Id2 restrains CD8+ T cell memory differentiation by inhibiting E2A-mediated direct activation of Tcf7 and that Id2 expression level mirrors T cell memory recall capacity. As a result of the defective effector differentiation, Id2-deficient CD8+ T cells fail to induce sufficient Tbx21 expression to generate short-lived effector CD8+ T cells. Our findings reveal that the Id2/E2A axis orchestrates T cell differentiation through the induction or repression of downstream transcription factors essential for effector and memory T cell differentiation. PMID:23536629

  19. Fibroblast growth factor represses Smad-mediated myofibroblast activation in aortic valvular interstitial cells

    PubMed Central

    Cushing, Melinda C.; Mariner, Peter D.; Liao, Jo-Tsu; Sims, Evan A.; Anseth, Kristi S.

    2008-01-01

    This study aimed to identify signaling pathways that oppose connective tissue fibrosis in the aortic valve. Using valvular interstitial cells (VICs) isolated from porcine aortic valve leaflets, we show that basic fibroblast growth factor (FGF-2) effectively blocks transforming growth factor-β1 (TGF-β1)-mediated myofibroblast activation. FGF-2 prevents the induction of α-smooth muscle actin (αSMA) expression and the exit of VICs from the cell cycle, both of which are hallmarks of myofibroblast activation. By blocking the activity of the Smad transcription factors that serve as the downstream nuclear effectors of TGF-β1, FGF-2 treatment inhibits fibrosis in VICs. Using an exogenous Smad-responsive transcriptional promoter reporter, we show that Smad activity is repressed by FGF-2, likely an effect of the fact that FGF-2 treatment prevents the nuclear localization of Smads in these cells. This appears to be a direct effect of FGF signaling through mitogen-activated protein kinase (MAPK) cascades as the treatment of VICs with the MAPK/extracellular regulated kinase (MEK) inhibitor U0126 acted to induce fibrosis and blocked the ability of FGF-2 to inhibit TGF-β1 signaling. Furthermore, FGF-2 treatment of VICs blocks the development of pathological contractile and calcifying phenotypes, suggesting that these pathways may be utilized in the engineering of effective treatments for valvular disease.—Cushing, M. C., Mariner, P. D., Liao, J. T., Sims, E. A., Anseth, K. S. Fibroblast growth factor represses Smad-mediated myofibroblast activation in aortic valvular interstitial cells. PMID:18218921

  20. 15-Deoxy-Δ(12,14)-prostaglandin J2 exerts pro- and anti-inflammatory effects in mesangial cells in a concentration-dependent manner.

    PubMed

    Martínez, Alma E; Sánchez-Gómez, Francisco J; Díez-Dacal, Beatriz; Oeste, Clara L; Pérez-Sala, Dolores

    2012-02-01

    Cyclopentenone prostaglandins play a modulatory role in inflammation, in part through their ability to covalently modify key proinflammatory proteins. Using mesangial cells as a cellular model of inflammation we have observed that 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) exerts a biphasic effect on cell activation by cytokines, with nanomolar concentrations eliciting an amplification of nitric oxide (NO) production and iNOS and COX-2 levels, and concentrations of 5 μM and higher inhibiting proinflammatory gene expression. An analog of 15d-PGJ(2) lacking the cyclopentenone structure (9,10-dihydro-15d-PGJ(2)) showed reduced ability to elicit both types of effects, suggesting that the electrophilic nature of 15d-PGJ(2) is important for its biphasic action. Interestingly, the switch from stimulatory to inhibitory actions occurred within a narrow concentration range and correlated with the ability of 15d-PGJ(2) to induce heme oxygenase 1 and γ-GCSm expression. These events are highly dependent on the triggering of the antioxidant response, which is considered as a sensor of thiol group modification. Indeed, the levels of the master regulator of the antioxidant response Nrf2 increased upon treatment with concentrations of 15d-PGJ(2) above 5 μM, an effect that could not be mimicked by 9,10-dihydro-15d-PGJ(2). Thus, an interplay of redox and electrophilic signalling mechanisms can be envisaged by which 15d-PGJ(2), as several other redox mediators, could contribute both to the onset and to the resolution of inflammation in a context or concentration-dependent manner.

  1. 15-Deoxy-Delta-12,14-Prostaglandin J2 Inhibits Lung Inflammation and Remodeling in Distinct Murine Models of Asthma

    PubMed Central

    Coutinho, Diego S.; Anjos-Valotta, Edna A.; do Nascimento, Caio V. M. F.; Pires, Ana Lucia A.; Napimoga, Marcelo H.; Carvalho, Vinícius F.; Torres, Rafael C.; e Silva, Patrícia M. R.; Martins, Marco A.

    2017-01-01

    15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) has been described as an anti-inflammatory lipid mediator in several in vitro and in vivo studies, but its effect on allergic pulmonary inflammation remains elusive. The aim of this study was to investigate the therapeutic potential of 15d-PGJ2 based on distinct murine models of allergic asthma triggered by either ovalbumin (OVA) or house dust mite extract (HDM). Characteristics of lung inflammation, airway hyper-reactivity (AHR), mucus exacerbation, and lung remodeling in sensitized A/J mice treated or not with 15d-PGJ2 were assessed. 15d-PGJ2 treatments were carried out systemically or topically given via subcutaneous injection or intranasal instillation, respectively. Analyses were carried out 24 h after the last allergen provocation. Irrespective of the route of administration, 15d-PGJ2 significantly inhibited the peribronchial accumulation of eosinophils and neutrophils, subepithelial fibrosis and also mucus exacerbation caused by either OVA or HDM challenge. The protective effect of 15d-PGJ2 occurred in parallel with inhibition of allergen-induced AHR and lung tissue production of pro-inflammatory cytokines, such as interleukin (IL)-5, IL-13, IL-17, and TNF-α. Finally, 15d-PGJ2 was found effective in inhibiting NF-κB phosphorylation upon HDM challenge as measured by Western blotting. In conclusion, our findings suggest that 15d-PGJ2 can reduce crucial features of asthma, including AHR, lung inflammation, and remodeling in distinct murine models of the disease. These effects are associated with a decrease in lung tissue generation of pro-inflammatory cytokines by a mechanism related to downregulation of NF-κB phosphorylation. PMID:28713373

  2. Glucocorticoid receptor and histone deacetylase-2 mediate dexamethasone-induced repression of MUC5AC gene expression.

    PubMed

    Chen, Yajun; Watson, Alan M; Williamson, Chad D; Rahimi, Michael; Liang, Chong; Colberg-Poley, Anamaris M; Rose, Mary C

    2012-11-01

    Airway occlusion in obstructive airway diseases is caused in part by the overproduction of secretory mucin glycoproteins through the up-regulation of mucin (MUC) genes by inflammatory mediators. Some pharmacological agents, including the glucocorticoid dexamethasone (Dex), repress mucin concentrations in lung epithelial cancer cells. Here, we show that Dex reduces the expression of MUC5AC, a major airway mucin gene, in primary differentiated normal human bronchial epithelial (NHBE) cells in a dose-dependent and time-dependent manner, and that the Dex-induced repression is mediated by the glucocorticoid receptor (GR) and two glucocorticoid response elements (GREs) in the MUC5AC promoter. The pre-exposure of cells to RU486, a GR antagonist, and mutations in either the GRE3 or GRE5 cis-sites abolished the Dex-induced repression. Chromatin immunoprecipitation (ChIP) assays showed a rapid temporal recruitment of GR to the GRE3 and GRE5 cis-elements in the MUC5AC promoter in NHBE and in A549 cells. Immunofluorescence showed nuclear colocalization of GR and histone deacetylase-2 (HDAC2) in MUC5AC-expressing NHBE cells. ChIP also showed a rapid temporal recruitment of HDAC2 to the GRE3 and GRE5 cis-elements in the MUC5AC promoter in both cell types. The knockdown of HDAC2 by HDAC2-specific short interfering RNA prevented the Dex-induced repression of MUC5AC in NHBE and A549 cells. These data demonstrate that GR and HDAC2 are recruited to the GRE3 and GRE5 cis-sites in the MUC5AC promoter and mediate the Dex-induced cis repression of MUC5AC gene expression. A better understanding of the mechanisms whereby glucocorticoids repress MUC5AC gene expression may be useful in formulating therapeutic interventions in chronic lung diseases.

  3. Repression of retrovirus-mediated transgene expression by interferons: implications for gene therapy.

    PubMed Central

    Ghazizadeh, S; Carroll, J M; Taichman, L B

    1997-01-01

    Retrovirus-mediated gene transfer is commonly used in gene therapy protocols and has the potential to provide long-term expression of the transgene. Although expression of a retrovirus-delivered transgene is satisfactory in cultured cells, it has been difficult to achieve consistent and high-level expression in vivo. In this investigation, we explored the possibility of modulating transgene expression by host-derived cytokines. Normal human keratinocytes and dermal fibroblasts were transduced with recombinant retroviruses expressing a reporter gene (lacZ). Treatment of transduced cells with a proinflammatory cytokine, gamma interferon (IFN-gamma), significantly reduced lacZ expression to less than 25% of that of nontreated cells. The inhibition was concentration dependent (peak at 5 ng/ml) and time dependent (maximal at 16 h for transcript and 24 h for protein); expression remained repressed in the continued presence of IFN-gamma but returned to normal levels 24 h after IFN-gamma withdrawal. The decrease in beta-galactosidase activity appeared to result from decrease in steady-state lacZ mRNA levels. Inhibitors of transcription and translation blocked IFN-gamma-induced repression, suggesting involvement of newly synthesized protein intermediates. Similar results were obtained by treatment of transduced cells with IFN-alpha but not with other proinflammatory cytokines, including tumor necrosis factor alpha, interleukin-2 (IL-1), IL-4, and granulocyte colony-stimulating factor. Although the level of lacZ mRNA was reduced by >70% following IFN treatment, the rate of lacZ transcription was not significantly different from that for nontreated cells. These results suggest that IFN-mediated regulation of transgene expression is at a posttranscriptional level. Interestingly, IFN-gamma also suppressed transgene expression driven by a cellular promoter (involucrin) inserted in an internal position in the retroviral vector. The presence of the overlapping 3' untranslated

  4. The Stress Response Mediator ATF3 Represses Androgen Signaling by Binding the Androgen Receptor

    PubMed Central

    Wang, Hongbo; Jiang, Ming; Cui, Hongmei; Chen, Mengqian; Buttyan, Ralph; Hayward, Simon W.; Hai, Tsonwin; Wang, Zhengxin

    2012-01-01

    Activating transcription factor 3 (ATF3) is a common mediator of cellular stress response signaling and is often aberrantly expressed in prostate cancer. We report here that ATF3 can directly bind the androgen receptor (AR) and consequently repress AR-mediated gene expression. The ATF3-AR interaction requires the leucine zipper domain of ATF3 that independently binds the DNA-binding and ligand-binding domains of AR, and the interaction prevents AR from binding to cis-acting elements required for expression of androgen-dependent genes while inhibiting the AR N- and C-terminal interaction. The functional consequences of the loss of ATF3 expression include increased transcription of androgen-dependent genes in prostate cancer cells that correlates with increased ability to grow in low-androgen-containing medium and increased proliferative activity of the prostate epithelium in ATF3 knockout mice that is associated with prostatic hyperplasia. Our results thus demonstrate that ATF3 is a novel repressor of androgen signaling that can inhibit AR functions, allowing prostate cells to restore homeostasis and maintain integrity in the face of a broad spectrum of intrinsic and environmental insults. PMID:22665497

  5. STAT4-mediated transcriptional repression of the IL5 gene in human memory Th2 cells.

    PubMed

    Gonzales-van Horn, Sarah R; Estrada, Leonardo D; van Oers, Nicolai S C; Farrar, J David

    2016-06-01

    Type I interferon (IFN-α/β) plays a critical role in suppressing viral replication by driving the transcription of hundreds of interferon-sensitive genes (ISGs). While many ISGs are transcriptionally activated by the ISGF3 complex, the significance of other signaling intermediates in IFN-α/β-mediated gene regulation remains elusive, particularly in rare cases of gene silencing. In human Th2 cells, IFN-α/β signaling suppressed IL5 and IL13 mRNA expression during recall responses to T-cell receptor (TCR) activation. This suppression occurred through a rapid reduction in the rate of nascent transcription, independent of de novo expression of ISGs. Further, IFN-α/β-mediated STAT4 activation was required for repressing the human IL5 gene, and disrupting STAT4 dimerization reversed this effect. This is the first demonstration of STAT4 acting as a transcriptional repressor in response to IFN-α/β signaling and highlights the unique activity of this cytokine to acutely block the expression of an inflammatory cytokine in human T cells.

  6. 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.

  7. Malate-Mediated Carbon Catabolite Repression in Bacillus subtilis Involves the HPrK/CcpA Pathway ▿ §

    PubMed Central

    Meyer, Frederik M.; Jules, Matthieu; Mehne, Felix M. P.; Le Coq, Dominique; Landmann, Jens J.; Görke, Boris; Aymerich, Stéphane; Stülke, Jörg

    2011-01-01

    Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate. PMID:22001508

  8. ChREBP mediates glucose repression of peroxisome proliferator-activated receptor alpha expression in pancreatic beta-cells.

    PubMed

    Boergesen, Michael; Poulsen, Lars la Cour; Schmidt, Søren Fisker; Frigerio, Francesca; Maechler, Pierre; Mandrup, Susanne

    2011-04-15

    Chronic exposure to elevated levels of glucose and fatty acids leads to dysfunction of pancreatic β-cells by mechanisms that are only partly understood. The transcription factor peroxisome proliferator-activated receptor α (PPARα) is an important regulator of genes involved in fatty acid metabolism and has been shown to protect against lipid-induced β-cell dysfunction. We and others have previously shown that expression of the PPARα gene in β-cells is rapidly repressed by glucose. Here we show that the PPARα gene is transcribed from five alternative transcription start sites, resulting in three alternative first exons that are spliced to exon 2. Expression of all PPARα transcripts is repressed by glucose both in insulinoma cells and in isolated pancreatic islets. The observation that the dynamics of glucose repression of PPARα transcription are very similar to those of glucose activation of target genes by the carbohydrate response element-binding protein (ChREBP) prompted us to investigate the potential role of ChREBP in the regulation of PPARα expression. We show that a constitutively active ChREBP lacking the N-terminal domain efficiently represses PPARα expression in insulinoma cells and in rodent and human islets. In addition, we demonstrate that siRNA-mediated knockdown of ChREBP abrogates glucose repression of PPARα expression as well as induction of well established ChREBP target genes in insulinoma cells. In conclusion, this work shows that ChREBP is a critical and direct mediator of glucose repression of PPARα gene expression in pancreatic β-cells, suggesting that ChREBP may be important for glucose suppression of the fatty acid oxidation capacity of β-cells.

  9. 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

  10. Amino acid 1-209 is essential for PDX-1-mediated repression of human CMV IE promoter activity.

    PubMed

    Chen, Jing; Chen, Lei; Li, Ge; Cheng, Lu; Huang, Yin; Zhang, Jia-xin; Fan, Wei-wei; Lu, Da-ru

    2006-11-01

    To explore the different roles of pancreatic duodenal homeobox factors-1 (PDX-1) domains in PDX-1 mediated repression of human cytomegalovirus immediately early (CMV IE) promoter. A series of truncated PDX-1 mutants were constructed. The binding of PDX-1 and CMV IE promoter was identified by electrophoretic mobility shift assay (EMSA). The dual-reporter assay was applied to examine the repression activities of PDX-1 mutants on CMV IE promoter. In addition, RNAi technology was used to specifically knock down the endogenous PDX-1 expression. The reporter assay indicated that compared to the mock controls (pEGFP-N2), overexpression of PDX-1 resulted in a 41% decrease of CMV IE promoter activity in the 293 cells (P< 0.05) and 43% decrease in HeLa cells (P< 0.05), and the repression levels of various truncated mutants played on CMV IE promoter were different. Specific knock down of the endogenous PDX-1 expression significantly restored the activity of CMV IE promoter. EMSA demonstrated that domain 3 is necessary for nuclear localization and DNA binding activity of PDX-1. However, binding of PDX-1 alone to CMV IE promoter was not sufficient to inhibit its transcriptional activity, and other domains of PDX-1 presented were also required. Our data suggested that the DNA binding activity of PDX-1 domain 3 and the cooperative binding of PDX-1 domain 1/2 with other proteins were required for PDX-1 mediated repression of CMV IE promoter.

  11. Id2 complexes with the SNAG domain of Snai1 inhibiting Snai1-mediated repression of integrin β4.

    PubMed

    Chang, Cheng; Yang, Xiaofang; Pursell, Bryan; Mercurio, Arthur M

    2013-10-01

    The epithelial-mesenchymal transition (EMT) is a fundamental process that underlies development and cancer. Although the EMT involves alterations in the expression of specific integrins that mediate stable adhesion to the basement membrane, such as α6β4, the mechanisms involved are poorly understood. Here, we report that Snai1 inhibits β4 transcription by increasing repressive histone modification (trimethylation of histone H3 at K27 [H3K27Me3]). Surprisingly, Snai1 is expressed and localized in the nucleus in epithelial cells, but it does not repress β4. We resolved this paradox by discovering that Id2 complexes with the SNAG domain of Snai1 on the β4 promoter and constrains the repressive function of Snai1. Disruption of the complex by depleting Id2 resulted in Snai1-mediated β4 repression with a concomitant increase in H3K27Me3 modification on the β4 promoter. These findings establish a novel function for Id2 in regulating Snai1 that has significant implications for the regulation of epithelial gene expression.

  12. Spatiotemporal Uncoupling of MicroRNA-Mediated Translational Repression and Target RNA Degradation Controls MicroRNP Recycling in Mammalian Cells.

    PubMed

    Bose, Mainak; Barman, Bahnisikha; Goswami, Avijit; Bhattacharyya, Suvendra N

    2017-02-15

    MicroRNA (miRNA)-mediated repression controls expression of more than half of protein-coding genes in metazoan animals. Translation repression is associated with target mRNA degradation initiated by decapping and deadenylation of the repressed mRNAs. Earlier evidence suggests the endoplasmic reticulum (ER) as the site where microRNPs (miRNPs) interact with their targets before translation repression sets in, but the subcellular location of subsequent degradation of miRNA-repressed messages is largely unidentified. Here, we explore the subcellular distribution of essential components of degradation machineries of miRNA-targeted mRNAs. We have noted that interaction of target mRNAs with AGO2 protein on the ER precedes the relocalization of repressed messages to multivesicular bodies (MVBs). The repressed messages subsequently get deadenylated, lose their interaction with AGO2, and become decapped. Blocking maturation of endosomes to late endosome and MVBs by targeting the endosomal protein HRS uncouples miRNA-mediated translation repression from target RNA degradation. HRS is also targeted by the intracellular parasite Leishmania donovani, which curtails the HRS level in infected cells to prevent uncoupling of mRNA-AGO2 interaction, preventing degradation of translationally repressed messages, and thus stops recycling of miRNPs preengaged in repression. Copyright © 2017 American Society for Microbiology.

  13. A Single Let-7 MicroRNA Bypasses LIN28-Mediated Repression.

    PubMed

    Triboulet, Robinson; Pirouz, Mehdi; Gregory, Richard I

    2015-10-13

    Let-7 microRNAs (miRNAs) are critical regulators of animal development, stem cell differentiation, glucose metabolism, and tumorigenesis. Mammalian genomes contain 12 let-7 isoforms that suppress expression of a common set of target mRNAs. LIN28 proteins selectively block let-7 biogenesis in undifferentiated cells and in cancer. The current model for coordinate let-7 repression involves the LIN28 cold-shock domain (CSD) binding the terminal loop and the two CCHC-type zinc fingers recognizing a GGAG sequence motif in precursor let-7 (pre-let-7) RNAs. Here, we perform a systematic analysis of all let-7 miRNAs and find that a single let-7 family member, human let-7a-3 (and its murine ortholog let-7c-2), escapes LIN28-mediated regulation. Mechanistically, we find that the pre-let-7c-2 loop precludes LIN28A binding and regulation. These findings refine the current model of let-7 regulation by LIN28 proteins and have important implications for understanding the LIN28/let-7 axis in development and disease. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

    PubMed Central

    Horlbeck, Max A; Gilbert, Luke A; Villalta, Jacqueline E; Adamson, Britt; Pak, Ryan A; Chen, Yuwen; Fields, Alexander P; Park, Chong Yon; Corn, Jacob E; Kampmann, Martin; Weissman, Jonathan S

    2016-01-01

    We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites. DOI: http://dx.doi.org/10.7554/eLife.19760.001 PMID:27661255

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

    SciTech Connect

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

    2015-12-04

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

  16. Ligand-induced repression of the glucocorticoid receptor gene is mediated by an NCoR1 repression complex formed by long-range chromatin interactions with intragenic glucocorticoid response elements.

    PubMed

    Ramamoorthy, Sivapriya; Cidlowski, John A

    2013-05-01

    Glucocorticoids are among the most potent and effective agents for treating inflammatory diseases and hematological cancers. However, subpopulations of patients are often resistant to steroid therapy, and determining the molecular mechanisms that contribute to glucocorticoid resistance is thus critical to addressing this clinical problem affecting patients with chronic inflammatory disorders. Since the cellular level of the glucocorticoid receptor (GR) is a critical determinant of glucocorticoid sensitivity and resistance, we investigated the molecular mechanisms mediating repression of glucocorticoid receptor gene expression. We show here that glucocorticoid-induced repression of GR gene expression is mediated by inhibition of transcription initiation. This process is orchestrated by the recruitment of agonist-bound GR to exon 6, followed by the assembly of a GR-NCoR1-histone deacetylase 3-containing repression complex at the transcriptional start site of the GR gene. A functional negative glucocorticoid response element (nGRE) in exon 6 of the GR gene and a long-range interaction occurring between this intragenic response element and the transcription start site appear to be instrumental in this repression. This autoregulatory mechanism of repression implies that the GR concentration can coordinate repression with excess ligand, regardless of the combinatorial associations of tissue-specific transcription factors. Consequently, the chronic nature of inflammatory conditions involving long-term glucocorticoid administration may lead to constitutive repression of GR gene transcription and thus to glucocorticoid resistance.

  17. Borna Disease Virus Phosphoprotein Represses p53-Mediated Transcriptional Activity by Interference with HMGB1

    PubMed Central

    Zhang, Guoqi; Kobayashi, Takeshi; Kamitani, Wataru; Komoto, Satoshi; Yamashita, Makiko; Baba, Satoko; Yanai, Hideyuki; Ikuta, Kazuyoshi; Tomonaga, Keizo

    2003-01-01

    Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that has a broad host range in warm-blooded animals, probably including humans. Recently, it was demonstrated that a 24-kDa phosphoprotein (P) of BDV directly binds to a multifunctional protein, amphoterin-HMGB1, and inhibits its function in cultured neural cells (W. Kamitani, Y. Shoya, T. Kobayashi, M. Watanabe, B. J. Lee, G. Zhang, K. Tomonaga, and K. Ikuta, J. Virol. 75:8742-8751, 2001). This observation suggested that expression of BDV P may cause deleterious effects in cellular functions by interference with HMGB1. In this study, we further investigated the significance of the binding between P and HMGB1. We demonstrated that P directly binds to the A-box domain on HMGB1, which is also responsible for interaction with a tumor suppression factor, p53. Recent works have demonstrated that binding between HMGB1 and p53 enhances p53-mediated transcriptional activity. Thus, we examined whether BDV P affects the transcriptional activity of p53 by interference with HMGB1. Mammalian two-hybrid analysis revealed that p53 and P competitively interfere with the binding of each protein to HMGB1 in a p53-deficient cell line, NCI-H1299. In addition, P was able to significantly decrease p53-mediated transcriptional activation of the cyclin G promoter. Furthermore, we showed that activation of p21waf1 expression was repressed in cyclosporine-treated BDV-infected cells, as well as p53-transduced NCI-H1299 cells. These results suggested that BDV P may be a unique inhibitor of p53 activity via binding to HMGB1. PMID:14581561

  18. Defining the Role of Post-Translational Modifications in SRC-3-Mediated Repression of mRNA Translation

    DTIC Science & Technology

    2012-10-01

    spectrometry from HeLa cells. The mutations that I have made for each amino acid are indicated. Specific Aim 1: Define if specific PTMs affect SRC-3...phosphorylation of these six key ‘transcriptional AA Residue/ Mutation Modification S 32A/E Phosphorylation S 214A Phosphorylation T 223A... mutation of these phosphorylation sites (SRC-31-6A) enhances SRC-3-mediated translational repression (Fig. 2A). Additionally, mutation of SRC-3

  19. Recruitment of Histone Methyltransferase G9a Mediates Transcriptional Repression of Fgf21 Gene by E4BP4 Protein*

    PubMed Central

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

    2013-01-01

    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

  20. 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.

  1. 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.

  2. Salt Stress Represses Soybean Seed Germination by Negatively Regulating GA Biosynthesis While Positively Mediating ABA Biosynthesis.

    PubMed

    Shu, Kai; Qi, Ying; Chen, Feng; Meng, Yongjie; Luo, Xiaofeng; Shuai, Haiwei; Zhou, Wenguan; Ding, Jun; Du, Junbo; Liu, Jiang; Yang, Feng; Wang, Qiang; Liu, Weiguo; Yong, Taiwen; Wang, Xiaochun; Feng, Yuqi; Yang, Wenyu

    2017-01-01

    Soybean is an important and staple oilseed crop worldwide. Salinity stress has adverse effects on soybean development periods, especially on seed germination and post-germinative growth. Improving seed germination and emergence will have positive effects under salt stress conditions on agricultural production. Here we report that NaCl delays soybean seed germination by negatively regulating gibberellin (GA) while positively mediating abscisic acid (ABA) biogenesis, which leads to a decrease in the GA/ABA ratio. This study suggests that fluridone (FLUN), an ABA biogenesis inhibitor, might be a potential plant growth regulator that can promote soybean seed germination under saline stress. Different soybean cultivars, which possessed distinct genetic backgrounds, showed a similar repressed phenotype during seed germination under exogenous NaCl application. Biochemical analysis revealed that NaCl treatment led to high MDA (malondialdehyde) level during germination and the post-germinative growth stages. Furthermore, catalase, superoxide dismutase, and peroxidase activities also changed after NaCl treatment. Subsequent quantitative Real-Time Polymerase Chain Reaction analysis showed that the transcription levels of ABA and GA biogenesis and signaling genes were altered after NaCl treatment. In line with this, phytohormone measurement also revealed that NaCl considerably down-regulated active GA1, GA3, and GA4 levels, whereas the ABA content was up-regulated; and therefore ratios, such as GA1/ABA, GA3/ABA, and GA4/ABA, are decreased. Consistent with the hormonal quantification, FLUN partially rescued the delayed-germination phenotype caused by NaCl-treatment. Altogether, these results demonstrate that NaCl stress inhibits soybean seed germination by decreasing the GA/ABA ratio, and that FLUN might be a potential plant growth regulator that could promote soybean seed germination under salinity stress.

  3. Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53.

    PubMed

    Lawhorn, Ingrid E B; Ferreira, Joshua P; Wang, Clifford L

    2014-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats) platform has been developed as a general method to direct proteins of interest to gene targets. While the native CRISPR system delivers a nuclease that cleaves and potentially mutates target genes, researchers have recently employed catalytically inactive CRISPR-associated 9 nuclease (dCas9) in order to target and repress genes without DNA cleavage or mutagenesis. With the intent of improving repression efficiency in mammalian cells, researchers have also fused dCas9 with a KRAB repressor domain. Here, we evaluated different genomic sgRNA targeting sites for repression of TP53. The sites spanned a 200-kb distance, which included the promoter, transcript sequence, and regions flanking the endogenous human TP53 gene. We showed that repression up to 86% can be achieved with dCas9 alone (i.e., without use of the KRAB domain) by targeting the complex to sites near the TP53 transcriptional start site. This work demonstrates that efficient transcriptional repression of endogenous human genes can be achieved by the targeted delivery of dCas9. Yet, the efficiency of repression strongly depends on the choice of the sgRNA target site.

  4. Possible involvement of 15-deoxy-Δ(12,14) -prostaglandin J2 in the development of leptin resistance.

    PubMed

    Hosoi, Toru; Matsuzaki, Syu; Miyahara, Tsuyoshi; Shimizu, Kaori; Hasegawa, Yuki; Ozawa, Koichiro

    2015-05-01

    Obesity is a worldwide health problem that urgently needs to be solved. Leptin is an anti-obesity hormone that activates satiety signals to the brain. Evidence to suggest that leptin resistance is involved in the development of obesity is increasing; however, the molecular mechanisms involved remain unclear. We herein demonstrated that 15-deoxy-Δ(12,14) -prostaglandin J2 (15d-PGJ2 ) was involved in the development of leptin resistance. A treatment with 15d-PGJ2 inhibited the leptin-induced activation of signal transducer and activator of transcription 3 (STAT3) in neuronal cells (SH-SY5Y-Ob-Rb cells). Furthermore, the intracerebroventricular administration of 15d-PGJ2 reversed the inhibitory effects of leptin on food intake in rats. The peroxisome proliferator-activated receptor gamma (PPAR-γ) antagonist, GW9662, slightly reversed the inhibitory effects of 15d-PGJ2 on the leptin-induced activation of STAT3 in neuronal cells. The PPAR-γ agonist, rosiglitazone, also inhibited leptin-induced STAT3 phosphorylation. Therefore, the inhibitory effects of 15d-PGJ2 may be mediated through PPAR-γ. On the other hand, 15d-PGJ2 -induced leptin resistance may not be mediated by endoplasmic reticulum stress or suppressor of cytokine signaling 3. The results of the present study suggest that 15d-PGJ2 is a novel factor for the development of leptin resistance in obesity. Leptin resistance, an insensitivity to the actions of leptin, is involved in the development of obesity. Here, we found 15-deoxy-Δ(12,14) -prostaglandin J2 (15d-PGJ2 ) may be involved in the development of leptin resistance. The present results suggest that the 15d-PGJ2 may be a novel factor for the development of leptin resistance in obesity. 15d-PGJ2 , 15-Deoxy-Δ(12,14) -prostaglandin J2; STAT3, signal tranducer and activator of transcription 3.

  5. Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells.

    PubMed

    Malaviya, Abhita; Sylvester, Paul W

    2014-01-01

    Previous findings showed that the anticancer effects of combined γ -tocotrienol and peroxisome proliferator activated receptor γ (PPAR γ ) antagonist treatment caused a large reduction in PPAR γ expression. However, other studies suggest that the antiproliferative effects of γ -tocotrienol and/or PPAR γ antagonists are mediated, at least in part, through PPAR γ -independent mechanism(s). Studies were conducted to characterize the role of PPAR γ in mediating the effects of combined treatment of γ -tocotrienol with PPAR γ agonists or antagonists on the growth of PPAR γ negative +SA mammary cells and PPAR γ -positive and PPAR γ -silenced MCF-7 and MDA-MB-231 breast cancer cells. Combined treatment of γ -tocotrienol with PPAR γ antagonist decreased, while combined treatment of γ -tocotrienol with PPAR γ agonist increased, growth of all cancer cells. However, treatment with high doses of 15d-PGJ2, an endogenous natural ligand for PPAR γ , had no effect on cancer cell growth. Western blot and qRT-PCR studies showed that the growth inhibitory effects of combined γ -tocotrienol and PPAR γ antagonist treatment decreased cyclooxygenase (COX-2), prostaglandin synthase (PGDS), and prostaglandin D2 (PGD2) synthesis. In conclusion, the anticancer effects of combined γ -tocotrienol and PPAR γ antagonists treatment in PPAR γ negative/silenced breast cancer cells are mediated through PPAR γ -independent mechanisms that are associated with a downregulation in COX-2, PGDS, and PGD2 synthesis.

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

    PubMed Central

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

    2016-01-01

    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

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

    PubMed Central

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

    2016-01-01

    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

  8. Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30{sup II}-mediated repression of LTR transcriptional activity

    SciTech Connect

    Michael, Bindhu; Nair, Amrithraj M.; Datta, Antara; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D. . E-mail: lairmore.1@osu.edu

    2006-10-25

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

  9. 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.

  10. 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

  11. The tumor suppressor, parafibromin, mediates histone H3 K9 methylation for cyclin D1 repression.

    PubMed

    Yang, Yong-Jin; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung

    2010-01-01

    Parafibromin, a component of the RNA polymerase II-associated PAF1 complex, is a tumor suppressor linked to hyperparathyroidism-jaw tumor syndrome and sporadic parathyroid carcinoma. Parafibromin induces cell cycle arrest by repressing cyclin D1 via an unknown mechanism. Here, we show that parafibromin interacts with the histone methyltransferase, SUV39H1, and functions as a transcriptional repressor. The central region (128-227 amino acids) of parafibromin is important for both the interaction with SUV39H1 and transcriptional repression. Parafibromin associated with the promoter and coding regions of cyclin D1 and was required for the recruitment of SUV39H1 and the induction of H3 K9 methylation but not H3 K4 methylation. RNA interference analysis showed that SUV39H1 was critical for cyclin D1 repression. These data suggest that parafibromin plays an unexpected role as a repressor in addition to its widely known activity associated with transcriptional activation. Parafibromin as a part of the PAF1 complex might downregulate cyclin D1 expression by integrating repressive H3 K9 methylation during transcription.

  12. Characterization of a two-component regulatory system that regulates succinate-mediated catabolite repression in Sinorhizobium meliloti.

    PubMed

    Garcia, Preston P; Bringhurst, Ryan M; Arango Pinedo, Catalina; Gage, Daniel J

    2010-11-01

    When they are available, Sinorhizobium meliloti utilizes C(4)-dicarboxylic acids as preferred carbon sources for growth while suppressing the utilization of some secondary carbon sources such as α- and β-galactosides. The phenomenon of using succinate as the sole carbon source in the presence of secondary carbon sources is termed succinate-mediated catabolite repression (SMCR). Genetic screening identified the gene sma0113 as needed for strong SMCR when S. meliloti was grown in succinate plus lactose, maltose, or raffinose. sma0113 and the gene immediately downstream, sma0114, encode the proteins Sma0113, an HWE histidine kinase with five PAS domains, and Sma0114, a CheY-like response regulator lacking a DNA-binding domain. sma0113 in-frame deletion mutants show a relief of catabolite repression compared to the wild type. sma0114 in-frame deletion mutants overproduce polyhydroxybutyrate (PHB), and this overproduction requires sma0113. Sma0113 may use its five PAS domains for redox level or energy state monitoring and use that information to regulate catabolite repression and related responses.

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

    PubMed

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

    2014-07-03

    1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. Nucleosomal location of the STE6 TATA box and Mat alpha 2p-mediated repression.

    PubMed

    Patterton, H G; Simpson, R T

    1994-06-01

    It has been proposed that yeast MATa cell-specific genes are repressed in MAT alpha cells by the Mat alpha 2p repressor-directed placement of a nucleosome in a position that incorporates the TATA box of the MATa-specific gene close to the nucleosomal pseudodyad. In this study, we address this proposal directly with a series of plasmids designed to place the MATa-specific STE6 TATA box at different locations in a nucleosome and in the internucleosomal linker. These plasmids contain different lengths of synthetic random DNA between the Mat alpha 2p operator and the TATA box of the STE6 promoter, which is located upstream of a lacZ reporter gene in a multicopy plasmid. We show that in MAT alpha cells, a nucleosome is retained in an identical translational frame relative to the Mat alpha 2p operator in all the constructs investigated, irrespective of the sequence of the DNA wrapped onto the histone octamer. This result shows that the nucleosomal organization of the STE6 promoter in MAT alpha cells is not conferred by the sequence of the promoter itself. No expression of the lacZ reporter gene was detectable in MAT alpha cells in any of the constructs, even with the TATA box located in a short internucleosomal linker. These data indicate that repression of MATa-specific genes in MAT alpha cells does not require the precise translational placement of the TATA box close to the nucleosomal pseudodyad; the gene remains repressed when the TATA box is located within the investigated 250-bp region in the organized chromatin domain abutting the Mat alpha 2p operator in MAT alpha cells and may remain repressed with the TATA box located anywhere within this organized repression domain.

  15. 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

  16. 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

  17. 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.

  18. Inhibition of root meristem growth by cadmium involves nitric oxide-mediated repression of auxin accumulation and signalling in Arabidopsis.

    PubMed

    Yuan, Hong-Mei; Huang, Xi

    2016-01-01

    The root is the first plant organ to get in contact with the toxin cadmium (Cd), which is a widespread soil contaminant. Cd inhibits the growth of the primary root, but the mechanisms underlying this inhibition remain elusive. In this study, we used physiological, pharmacological and genetic approaches to investigate the roles of nitric oxide (NO) and auxin in Cd-mediated inhibition of Arabidopsis thaliana root meristem growth. Our study demonstrated that in the first 12 h of exposure, Cd inhibits primary root elongation through a decrease in the sizes of both the elongation and meristematic zones. Following Cd exposure, a decrease in auxin levels is associated with reduced PIN1/3/7 protein accumulation, but not with reduced PIN1/3/7 transcript levels. Additionally, Cd stabilized AXR3/IAA17 protein to repress auxin signalling in this Cd-mediated process. Furthermore, decreasing Cd-induced NO accumulation with either NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N(ω) -nitro-l-Arg-methylester (l-NAME) compromised the Cd-mediated inhibition of root meristem development, reduction in auxin and PIN1/3/7 accumulation, as well as stabilization of AXR3/IAA17, indicating that NO participates in Cd-mediated inhibition of root meristem growth. Taken together, our data suggest that Cd inhibits root meristem growth by NO-mediated repression of auxin accumulation and signalling in Arabidopsis. © 2015 John Wiley & Sons Ltd.

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

    PubMed Central

    Farmer, Ryan M.

    2015-01-01

    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

  20. The zinc finger and C-terminal domains of MTA proteins are required for FOG-2-mediated transcriptional repression via the NuRD complex.

    PubMed

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

    2008-02-01

    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. Furthermore, 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.

  1. Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene.

    PubMed

    Hadjighassem, Mahmoud R; Galaraga, Kimberly; Albert, Paul R

    2011-01-01

    The serotonin-1A (5-HT1A) receptor functions as a pre-synaptic autoreceptor in serotonin neurons that regulates their activity, and is also widely expressed on non-serotonergic neurons as a post-synaptic heteroreceptor to mediate serotonin action. The 5-HT1A receptor gene is strongly repressed by a dual repressor element (DRE), which is recognized by two proteins: Freud-1/CC2D1A and another unknown protein. Here we identify mouse Freud-2/CC2D1B as the second repressor of the 5-HT1A-DRE. Freud-2 shares 50% amino acid identity with Freud-1, and contains conserved structural domains. Mouse Freud-2 bound specifically to the rat 5-HT1A-DRE adjacent to, and partially overlapping, the Freud-1 binding site. By supershift assay using nuclear extracts from L6 myoblasts, Freud-2-DRE complexes were distinguished from Freud-1-DRE complexes. Freud-2 mRNA and protein were detected throughout mouse brain and peripheral tissues. Freud-2 repressed 5-HT1A promoter-reporter constructs in a DRE-dependent manner in non-neuronal (L6) or 5-HT1A-expressing neuronal (NG108-15, RN46A) cell models. In NG108-15 cells, knockdown of Freud-2 using a specific short-interfering RNA reduced endogenous Freud-2 protein levels and decreased Freud-2 bound to the 5-HT1A-DRE as detected by chromatin immunoprecipitation assay, but increased 5-HT1A promoter activity and 5-HT1A protein levels. Taken together, these data show that Freud-2 is the second component that, with Freud-1, mediates dual repression of the 5-HT1A receptor gene at the DRE.

  2. Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene

    PubMed Central

    Hadjighassem, Mahmoud R.; Galaraga, Kimberly; Albert, Paul R.

    2015-01-01

    The serotonin-1A (5-HT1A) receptor functions as a pre-synaptic autoreceptor in serotonin neurons that regulates their activity, and is also widely expressed on non-serotonergic neurons as a post-synaptic heteroreceptor to mediate serotonin action. The 5-HT1A receptor gene is strongly repressed by a dual repressor element (DRE), which is recognized by two proteins: Freud-1/CC2D1A and another unknown protein. Here we identify mouse Freud-2/CC2D1B as the second repressor of the 5-HT1A–DRE. Freud-2 shares 50% amino acid identity with Freud-1, and contains conserved structural domains. Mouse Freud-2 bound specifically to the rat 5-HT1A–DRE adjacent to, and partially overlapping, the Freud-1 binding site. By supershift assay using nuclear extracts from L6 myoblasts, Freud-2–DRE complexes were distinguished from Freud-1–DRE complexes. Freud-2 mRNA and protein were detected throughout mouse brain and peripheral tissues. Freud-2 repressed 5-HT1A promoter–reporter constructs in a DRE-dependent manner in non-neuronal (L6) or 5-HT1A-expressing neuronal (NG108-15, RN46A) cell models. In NG108-15 cells, knockdown of Freud-2 using a specific short-interfering RNA reduced endogenous Freud-2 protein levels and decreased Freud-2 bound to the 5-HT1A–DRE as detected by chromatin immunoprecipitation assay, but increased 5-HT1A promoter activity and 5-HT1A protein levels. Taken together, these data show that Freud-2 is the second component that, with Freud-1, mediates dual repression of the 5-HT1A receptor gene at the DRE. PMID:21155902

  3. 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.

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

    PubMed Central

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

    2016-01-01

    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

  5. Could repressive coping be a mediating factor in the symptom profile of individuals diagnosed with schizophrenia?

    PubMed

    Scholes, B; Martin, C R

    2010-06-01

    Despite a relatively high prevalence, and the enduring patronage of the disorder by psychiatry and the pharmaceutical industry, innovative conceptualization of schizophrenia in a client-empowering and quality of life-enhancing way appears to represent a vacuum within the clinical agenda, certainly taking second place to 'patient management'. However, against this bland background of medicalization of what is clearly a poorly understood and complex multifactorial syndrome, innovative treatment approaches aimed at symptom control, in particular, the stress vulnerability model (SVM), have been developed. However, the SVM is an incomplete model of patient experience and says little of aetiological note. One area of psychological function that may give further insight into the symptom experience associated with schizophrenia within the context of stress vulnerability concerns the mechanisms of repression. Ironically, the notion of repression will for many represent the epitome of nonevidence-based psychiatric theory and related psychodynamic therapy practice. However, more contemporary work within the psychological literature has aimed to make the concept both measurable and observable. No longer occluded by the context of psychoanalysis, cognitive science accounts of repression may be of value in facilitating understanding of the variability and predictability of symptoms of schizophrenia and may provide a dimension of therapeutic engagement allied to the SVM.

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

    PubMed Central

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

    2016-01-01

    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

  7. 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.

  8. The RING domain of Mdm2 mediates histone ubiquitylation and transcriptional repression.

    PubMed

    Minsky, Neri; Oren, Moshe

    2004-11-19

    Histone modifications play a pivotal role in regulating transcription and other chromatin-associated processes. In yeast, histone H2B monoubiquitylation affects gene silencing. However, mammalian histone ubiquitylation remains poorly understood. We report that the Mdm2 oncoprotein, a RING domain E3 ubiquitin ligase known to ubiquitylate the p53 tumor suppressor protein, can interact directly with histones and promote in vitro monoubiquitylation of histones H2A and H2B. Moreover, Mdm2 induces H2B monoubiquitylation in vivo. Endogenous Mdm2 is tethered in vivo, presumably via p53, to chromatin comprising the p53-responsive p21(waf1) promoter, and Mdm2 overexpression enhances protein ubiquitylation in the vicinity of a p53 binding site within that promoter. Moreover, when recruited to a promoter in the absence of p53, Mdm2 can repress transcription dependently on its RING domain, suggesting that its E3 activity contributes to repression. Histone ubiquitylation may thus constitute a novel mechanism of transcriptional repression by Mdm2, possibly underlying some of its oncogenic activities.

  9. 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

  10. 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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Telomere-Mediated Plasmid Segregation in Saccharomyces Cerevisiae Involves Gene Products Required for Transcriptional Repression at Silencers and Telomeres

    PubMed Central

    Longtine, M. S.; Enomoto, S.; Finstad, S. L.; Berman, J.

    1993-01-01

    Plasmids that contain Saccharomyces cerevisiae TG(1-3) telomere repeat sequences (TRS plasmids) segregate efficiently during mitosis. Mutations in histone H4 reduce the efficiency of TRS-mediated plasmid segregation, suggesting that chromatin structure is involved in this process. Sir2, Sir3 and Sir4 are required for the transcriptional repression of genes located at the silent mating type loci (HML and HMR) and at telomeres (telomere position effect) and are also involved in the segregation of TRS plasmids, indicating that TRS-mediated plasmid segregation involves factors that act at chromosomal telomeres. TRS plasmid segregation differs from the segregation of plasmids carrying the HMR E silencing region: HMR E plasmid segregation function is completely dependent upon Sir2, Sir3 and Sir4, involves Sir1 and is not influenced by mutations in RAP1 that eliminate TRS plasmid segregation. Mutations in SIR1, SIN1, TOP1, TEL1 and TEL2 do not influence TRS plasmid segregation. Unlike transcriptional repression at telomeres, TRS plasmids retain partial segregation function in sir2, sir3, sir4, nat1 and ard1 mutant strains. Thus it is likely that TRS plasmid segregation involves additional factors that are not involved in telomere position effect. PMID:8436267

  12. The Saccharomyces cerevisiae Cdk8 Mediator Represses AQY1 Transcription by Inhibiting Set1p-Dependent Histone Methylation

    PubMed Central

    Law, Michael J.; Finger, Michael A.

    2017-01-01

    In the budding yeast Saccharomyces cerevisiae, nutrient depletion induces massive transcriptional reprogramming that relies upon communication between transcription factors, post-translational histone modifications, and the RNA polymerase II holoenzyme complex. Histone H3Lys4 methylation (H3Lys4 me), regulated by the Set1p-containing COMPASS methyltransferase complex and Jhd2p demethylase, is one of the most well-studied histone modifications. We previously demonstrated that the RNA polymerase II mediator components cyclin C-Cdk8p inhibit locus-specific H3Lys4 3me independently of Jhd2p. Here, we identify loci subject to cyclin C- and Jhd2p-dependent histone H3Lys4 3me inhibition using chromatin immunoprecipitation (ChIP)-seq. We further characterized the independent and combined roles of cyclin C and Jhd2p in controlling H3Lys4 3me and transcription in response to fermentable and nonfermentable carbon at multiple loci. These experiments suggest that H3Lys4 3me alone is insufficient to induce transcription. Interestingly, we identified an unexpected role for cyclin C-Cdk8p in repressing AQY1 transcription, an aquaporin whose expression is normally induced during nutrient deprivation. These experiments, combined with previous work in other labs, support a two-step model in which cyclin C-Cdk8p mediate AQY1 transcriptional repression by stimulating transcription factor proteolysis and preventing Set1p recruitment to the AQY1 locus. PMID:28143948

  13. Noncanonical role of Arabidopsis COP1/SPA complex in repressing BIN2-mediated PIF3 phosphorylation and degradation in darkness.

    PubMed

    Ling, Jun-Jie; Li, Jian; Zhu, Danmeng; Deng, Xing Wang

    2017-03-28

    The E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) has been known to mediate key signaling factors for degradation via the ubiquitin/26S proteasome pathway in both plants and animals. Here, we report a noncanonical function of Arabidopsis COP1, the central repressor of photomorphogenesis, in the form of a COP1/ SUPPRESSOR of phyA-105 (SPA) complex. We show that the COP1/SPA complex associates with and stabilizes PHYTOCHROME INTERACTING FACTOR 3 (PIF3) to repress photomorphogenesis in the dark. We identify the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) as a kinase of PIF3, which induces PIF3 degradation via 26S proteasome during skotomorphogenesis. Mutations on two typical BIN2 phosphorylation motifs of PIF3 lead to a strong stabilization of the protein in the dark. We further show that the COP1/SPA complex promotes PIF3 stability by repressing BIN2 activity. Intriguingly, without affecting BIN2 expression, the COP1/SPA complex modulates BIN2 activity through interfering with BIN2-PIF3 interaction, thereby inhibiting BIN2-mediated PIF3 phosphorylation and degradation. Taken together, our results suggest another paradigm for COP1/SPA complex action in the precise control of skotomorphogenesis.

  14. Imperfect centered miRNA binding sites are common and can mediate repression of target mRNAs

    PubMed Central

    2014-01-01

    Background MicroRNAs (miRNAs) bind to mRNAs and target them for translational inhibition or transcriptional degradation. It is thought that most miRNA-mRNA interactions involve the seed region at the 5′ end of the miRNA. The importance of seed sites is supported by experimental evidence, although there is growing interest in interactions mediated by the central region of the miRNA, termed centered sites. To investigate the prevalence of these interactions, we apply a biotin pull-down method to determine the direct targets of ten human miRNAs, including four isomiRs that share centered sites, but not seeds, with their canonical partner miRNAs. Results We confirm that miRNAs and their isomiRs can interact with hundreds of mRNAs, and that imperfect centered sites are common mediators of miRNA-mRNA interactions. We experimentally demonstrate that these sites can repress mRNA activity, typically through translational repression, and are enriched in regions of the transcriptome bound by AGO. Finally, we show that the identification of imperfect centered sites is unlikely to be an artifact of our protocol caused by the biotinylation of the miRNA. However, the fact that there was a slight bias against seed sites in our protocol may have inflated the apparent prevalence of centered site-mediated interactions. Conclusions Our results suggest that centered site-mediated interactions are much more frequent than previously thought. This may explain the evolutionary conservation of the central region of miRNAs, and has significant implications for decoding miRNA-regulated genetic networks, and for predicting the functional effect of variants that do not alter protein sequence. PMID:24629056

  15. 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

    2016-02-02

    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

  16. HUB1, a novel Krüppel type zinc finger protein, represses the human T cell leukemia virus type I long terminal repeat-mediated expression.

    PubMed Central

    Okumura, K; Sakaguchi, G; Naito, K; Tamura, T; Igarashi, H

    1997-01-01

    We have shown that human T-cell leukemia virus type I (HTLV-I) gene expression is negatively regulated by the U5 repressive element (U5RE) of its long terminal repeat (LTR). To isolate factors binding to U5RE, we screened a cDNA expression library by south-western blotting with a U5RE probe. Screening 2 x10(6) clones gave a positive clone with a 3.8 kb insert encoding a novel 671 residue polypeptide, named HTLV-I U5RE binding protein 1 (HUB1), with five zinc finger domains and a Krüppel-associated box like domain; HUB1 may be related to a repressor belonging to the Krüppel type zinc finger protein. A 4.0 kb mRNA for HUB1 is ubiquitously expressed among all human tissues tested. HUB1 recognizes the TCCACCCC sequence as a core motif and exerts a strong repressive effect on HTLV-I LTR-mediated expression. A new repressive domain, named HUB1 repressive (HUR) domain, was identified, rather than the Krüppel-associated box like domain. The N-terminal region upstream of HUR domain seemed to be also indispensable to the repression. Thus, we propose that HUB1 is a new type repressor and plays an important role in the HTLV-I U5-mediated repression. PMID:9396811

  17. The mitotic Clb cyclins are required to alleviate HIR-mediated repression of the yeast histone genes at the G1/S transition

    PubMed Central

    Amin, Amit Dipak; Dimova, Dessislava K.; Ferreira, Monica E.; Vishnoi, Nidhi; Hancock, Leandria C.; Osley, Mary Ann; Prochasson, Philippe

    2011-01-01

    The histone genes are an important group of cell cycle regulated genes whose transcription is activated during the G1/S transition and repressed in early G1, late S, and G2/M. The HIR complex, comprised of Hir1, Hir2, Hir3 and Hpc2, regulates three of the four histone gene loci. While relief of repression at the G1/S boundary involves the HIR complex, as well as other cofactors, the mechanism by which this derepression occurs remains unknown. To better understand how transcriptional repression contributes to periodic expression in the cell cycle, we sought to identify the cell cycle signals required to alleviate HIR-mediated repression of the histone genes. By measuring histone gene transcription in strains with various combinations of clb mutations, we found that the mitotic Clb1/Clb2 cyclins are required to alleviate Hir-mediated repression during the G1/S transition and that Clb2 physically interacts with the HIR complex. While the HIR complex regulates histone genes transcription in combination with two other histone H3/H4 chaperones, Asf1 and Rtt106, our data demonstrate that the mitotic Clb cyclins are necessary to specifically alleviate the repressive action of the HIR complex itself in order to allow proper expression of the histone genes in late G1/early S phase. PMID:21978826

  18. Citrullination of Histone H3 Interferes with HP1-Mediated Transcriptional Repression

    PubMed Central

    Sharma, Priyanka; Azebi, Saliha; England, Patrick; Christensen, Tove; Møller-Larsen, Anné; Petersen, Thor; Batsché, Eric; Muchardt, Christian

    2012-01-01

    Multiple Sclerosis (MS) is an autoimmune disease associated with abnormal expression of a subset of cytokines, resulting in inappropriate T-lymphocyte activation and uncontrolled immune response. A key issue in the field is the need to understand why these cytokines are transcriptionally activated in the patients. Here, we have examined several transcription units subject to pathological reactivation in MS, including the TNFα and IL8 cytokine genes and also several Human Endogenous RetroViruses (HERVs). We find that both the immune genes and the HERVs require the heterochromatin protein HP1α for their transcriptional repression. We further show that the Peptidylarginine Deiminase 4 (PADI4), an enzyme with a suspected role in MS, weakens the binding of HP1α to tri-methylated histone H3 lysine 9 by citrullinating histone H3 arginine 8. The resulting de-repression of both cytokines and HERVs can be reversed with the PADI-inhibitor Cl-amidine. Finally, we show that in peripheral blood mononuclear cells (PBMCs) from MS patients, the promoters of TNFα, and several HERVs share a deficit in HP1α recruitment and an augmented accumulation of histone H3 with a double citrulline 8 tri-methyl lysine 9 modifications. Thus, our study provides compelling evidence that HP1α and PADI4 are regulators of both immune genes and HERVs, and that multiple events of transcriptional reactivation in MS patients can be explained by the deficiency of a single mechanism of gene silencing. PMID:23028349

  19. 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

  20. 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

    2015-05-11

    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.

  1. EZH2-mediated Puma gene repression regulates non-small cell lung cancer cell proliferation and cisplatin-induced apoptosis.

    PubMed

    Liu, Haidan; Li, Wei; Yu, Xinfang; Gao, Feng; Duan, Zhi; Ma, Xiaolong; Tan, Shiming; Yuan, Yunchang; Liu, Lijun; Wang, Jian; Zhou, Xinmin; Yang, Yifeng

    2016-08-30

    Polycomb group (PcG) proteins are highly conserved epigenetic effectors that maintain the silenced state of genes. EZH2 is the catalytic core and one of the most important components of the polycomb repressive complex 2 (PRC2). In non-small cell lung cancer (NSCLC) cells and primary lung tumors, we found that PRC2 components, including EZH2, are overexpressed. High levels of EZH2 protein were associated with worse overall survival rate in NSCLC patients. RNA interference mediated attenuation of EZH2 expression blunted the malignant phenotype in this setting, exerting inhibitory effects on cell proliferation, anchorage-independent growth, and tumor development in a xenograft mouse model. Unexpectedly, we discovered that, in the suppression of EZH2, p53 upregulated modulator of apoptosis (PUMA) expression was concomitantly induced. This is achieved through EZH2 directly binds to the Puma promoter thus epigenetic repression of PUMA expression. Furthermore, cisplatin-induced apoptosis of EZH2-knocking down NSCLC cells was elevated as a consequence of increased PUMA expression. Our work reveals a novel epigenetic regulatory mechanism controlling PUMA expression and suggests that EZH2 offers a candidate molecular target for NSCLC therapy and EZH2-regulated PUMA induction would synergistically increase the sensitivity to platinum agents in non-small cell lung cancers.

  2. EZH2-mediated Puma gene repression regulates non-small cell lung cancer cell proliferation and cisplatin-induced apoptosis

    PubMed Central

    Yu, Xinfang; Gao, Feng; Duan, Zhi; Ma, Xiaolong; Tan, Shiming; Yuan, Yunchang; Liu, Lijun; Wang, Jian; Zhou, Xinmin; Yang, Yifeng

    2016-01-01

    Polycomb group (PcG) proteins are highly conserved epigenetic effectors that maintain the silenced state of genes. EZH2 is the catalytic core and one of the most important components of the polycomb repressive complex 2 (PRC2). In non-small cell lung cancer (NSCLC) cells and primary lung tumors, we found that PRC2 components, including EZH2, are overexpressed. High levels of EZH2 protein were associated with worse overall survival rate in NSCLC patients. RNA interference mediated attenuation of EZH2 expression blunted the malignant phenotype in this setting, exerting inhibitory effects on cell proliferation, anchorage-independent growth, and tumor development in a xenograft mouse model. Unexpectedly, we discovered that, in the suppression of EZH2, p53 upregulated modulator of apoptosis (PUMA) expression was concomitantly induced. This is achieved through EZH2 directly binds to the Puma promoter thus epigenetic repression of PUMA expression. Furthermore, cisplatin-induced apoptosis of EZH2-knocking down NSCLC cells was elevated as a consequence of increased PUMA expression. Our work reveals a novel epigenetic regulatory mechanism controlling PUMA expression and suggests that EZH2 offers a candidate molecular target for NSCLC therapy and EZH2-regulated PUMA induction would synergistically increase the sensitivity to platinum agents in non-small cell lung cancers. PMID:27472460

  3. Convergent repression of miR156 by sugar and the CDK8 module of Arabidopsis Mediator.

    PubMed

    Buendía-Monreal, Manuel; Gillmor, C Stewart

    2017-03-01

    In Arabidopsis, leaves produced during the juvenile vegetative phase are simple, while adult leaves are morphologically complex. The juvenile to adult transition is regulated by miR156, a microRNA that promotes juvenility by impeding the function of SPL transcription factors, which specify adult leaf traits. Both leaf derived sugars, as well as the Mediator Cyclin Dependent Kinase 8 (CDK8) module genes CENTER CITY (CCT)/MED12 and GRAND CENTRAL (GCT)/MED13, act upstream of miR156 to promote the juvenile to adult transition. However, it is not known whether sugar, CCT and GCT repress miR156 independently, as part of the same pathway, or in a cooperative manner. Here we show that sugar treatment can repress MIR156 expression in the absence of CCT or GCT. Both cct and the photosynthetic mutant chlorina1 (ch1) (which decreases sugar synthesis) exhibit extended juvenile development and increased MIR156A and MIR156C expression. Compared to ch1 and cct single mutants, the ch1 cct double mutant has a stronger effect on juvenile leaf traits, higher MIR156C levels, and a dramatic increase in MIR156A. Our results show that sugar and the CDK8 module are capable of regulating MIR156 independently, but suggest they normally act together in a synergistic manner. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. α-Synuclein induces alterations in adult neurogenesis in Parkinson disease models via p53-mediated repression of Notch1.

    PubMed

    Desplats, Paula; Spencer, Brian; Crews, Leslie; Pathel, Pruthul; Morvinski-Friedmann, Dinorah; Kosberg, Kori; Roberts, Scott; Patrick, Christina; Winner, Beate; Winkler, Juergen; Masliah, Eliezer

    2012-09-14

    Parkinson disease is characterized by the loss of dopaminergic neurons mainly in the substantia nigra. Accumulation of α-synuclein and cell loss has been also reported in many other brain regions including the hippocampus, where it might impair adult neurogenesis, contributing to nonmotor symptoms. However, the molecular mechanisms of these alterations are still unknown. In this report we show that α-synuclein-accumulating adult rat hippocampus neural progenitors present aberrant neuronal differentiation, with reduction of Notch1 expression and downstream signaling targets. We characterized a Notch1 proximal promoter that contains p53 canonical response elements. In vivo binding of p53 represses the transcription of Notch1 in neurons. Moreover, we demonstrated that α-synuclein directly binds to the DNA at Notch1 promoter vicinity and also interacts with p53 protein, facilitating or increasing Notch1 signaling repression, which interferes with maturation and survival of neural progenitors cells. This study provides a molecular basis for α-synuclein-mediated disruption of adult neurogenesis in Parkinson disease.

  5. A Herpesviral induction of RAE-1 NKG2D ligand expression occurs through release of HDAC mediated repression

    PubMed Central

    Greene, Trever T; Tokuyama, Maria; Knudsen, Giselle M; Kunz, Michele; Lin, James; Greninger, Alexander L; DeFilippis, Victor R; DeRisi, Joseph L; Raulet, David H; Coscoy, Laurent

    2016-01-01

    Natural Killer (NK) cells are essential for control of viral infection and cancer. NK cells express NKG2D, an activating receptor that directly recognizes NKG2D ligands. These are expressed at low level on healthy cells, but are induced by stresses like infection and transformation. The physiological events that drive NKG2D ligand expression during infection are still poorly understood. We observed that the mouse cytomegalovirus encoded protein m18 is necessary and sufficient to drive expression of the RAE-1 family of NKG2D ligands. We demonstrate that RAE-1 is transcriptionally repressed by histone deacetylase inhibitor 3 (HDAC3) in healthy cells, and m18 relieves this repression by directly interacting with Casein Kinase II and preventing it from activating HDAC3. Accordingly, we found that HDAC inhibiting proteins from human herpesviruses induce human NKG2D ligand ULBP-1. Thus our findings indicate that virally mediated HDAC inhibition can act as a signal for the host to activate NK-cell recognition. DOI: http://dx.doi.org/10.7554/eLife.14749.001 PMID:27874833

  6. A Herpesviral induction of RAE-1 NKG2D ligand expression occurs through release of HDAC mediated repression.

    PubMed

    Greene, Trever T; Tokuyama, Maria; Knudsen, Giselle M; Kunz, Michele; Lin, James; Greninger, Alexander L; DeFilippis, Victor R; DeRisi, Joseph L; Raulet, David H; Coscoy, Laurent

    2016-11-22

    Natural Killer (NK) cells are essential for control of viral infection and cancer. NK cells express NKG2D, an activating receptor that directly recognizes NKG2D ligands. These are expressed at low level on healthy cells, but are induced by stresses like infection and transformation. The physiological events that drive NKG2D ligand expression during infection are still poorly understood. We observed that the mouse cytomegalovirus encoded protein m18 is necessary and sufficient to drive expression of the RAE-1 family of NKG2D ligands. We demonstrate that RAE-1 is transcriptionally repressed by histone deacetylase inhibitor 3 (HDAC3) in healthy cells, and m18 relieves this repression by directly interacting with Casein Kinase II and preventing it from activating HDAC3. Accordingly, we found that HDAC inhibiting proteins from human herpesviruses induce human NKG2D ligand ULBP-1. Thus our findings indicate that virally mediated HDAC inhibition can act as a signal for the host to activate NK-cell recognition.

  7. 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.

  8. Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes.

    PubMed

    Cheng, De; Zhao, Yuanjun; Wang, Shuwen; Zhang, Fan; Russo, Mariano; McMahon, Steven B; Zhu, Jiyue

    2017-03-01

    The human telomerase reverse transcriptase (hTERT) gene is repressed in most somatic cells, whereas the expression of the mouse mTert gene is widely detected. To understand the mechanisms of this human-specific repression, we constructed bacterial artificial chromosome (BAC) reporters using human and mouse genomic DNAs encompassing the TERT genes and neighboring loci. Upon chromosomal integration, the hTERT, but not the mTert, reporter was stringently repressed in telomerase-negative human cells in a histone deacetylase (HDAC)-dependent manner, replicating the expression of their respective endogenous genes. In chimeric BACs, the mTert promoter became strongly repressed in the human genomic context, but the hTERT promoter was highly active in the mouse genomic context. Furthermore, an unrelated herpes simplex virus-thymidine kinase (HSV-TK) promoter was strongly repressed in the human, but not in the mouse, genomic context. These results demonstrated that the repression of hTERT gene was dictated by distal elements and its chromatin environment. This repression depended on class I HDACs and involved multiple corepressor complexes, including HDAC1/2-containing Sin3B, nucleosome remodeling and histone deacetylase (NuRD), and corepressor of RE1 silencing transcription factor (CoREST) complexes. Together, our data indicate that the lack of telomerase expression in most human somatic cells results from its repressive genomic environment, providing new insight into the mechanism of long-recognized differential telomerase regulation in mammalian species.-Cheng, D., Zhao, Y., Wang, S., Zhang, F., Russo, M., McMahon, S. B., Zhu, J. Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes. © FASEB.

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

    PubMed

    Barman, Bahnisikha; Bhattacharyya, Suvendra N

    2015-10-09

    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.

  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. 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

  12. 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.

  13. 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. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Hepatic injury associated with Trypanosoma cruzi infection is attenuated by treatment with 15-deoxy-Δ(12,14) prostaglandin J2.

    PubMed

    Penas, Federico Nicolás; Cevey, Ágata Carolina; Siffo, Sofía; Mirkin, Gerardo Ariel; Goren, Nora Beatriz

    2016-11-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, causes an intense inflammatory response in several tissues, including the liver. Since this organ is central to metabolism, its infection may be reflected in the outcome of the disease. 15-deoxy-Δ(12,14) prostaglandin J2 (15dPGJ2), a natural agonist of peroxisome-proliferator activated receptor (PPAR) γ, has been shown to exert anti-inflammatory effects in the heart upon T. cruzi infection. However, its role in the restoration of liver function and reduction of liver inflammation has not been studied yet. BALB/c mice were infected with T. cruzi. The effects of in vivo treatment with 15dPGJ2 on liver inflammation and fibrosis, as well as on the GOT/GPT ratio were studied and the role of NF-κB pathway on 15dPGJ2-mediated effects was analysed. 15dPGJ2 reduced liver inflammatory infiltrates, proinflammatory enzymes and cytokines expression, restored the De Ritis ratio values to normal, reduced the deposits of interstitial and perisinusoidal collagen, reduced the expression of the pro-fibrotic cytokines and inhibited the translocation of the p65 NF-κB subunit to the nucleus. Thus, we showed that 15dPGJ2 is able to significantly reduce the inflammatory response and fibrosis and reduced enzyme markers of liver damage in mice infected with T. cruzi.

  15. Effect of peroxisome proliferator activated receptor (PPAR)gamma agonists on prostaglandins cascade in joint cells.

    PubMed

    Moulin, David; Poleni, Paul-Emile; Kirchmeyer, Mélanie; Sebillaud, Sylvie; Koufany, Meriem; Netter, Patrick; Terlain, Bernard; Bianchi, Arnaud; Jouzeau, Jean-Yves

    2006-01-01

    In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.

  16. 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.

  17. dbx mediates neuronal specification and differentiation through cross-repressive, lineage-specific interactions with eve and hb9.

    PubMed

    Lacin, Haluk; Zhu, Yi; Wilson, Beth A; Skeath, James B

    2009-10-01

    Individual neurons adopt and maintain defined morphological and physiological phenotypes as a result of the expression of specific combinations of transcription factors. In particular, homeodomain-containing transcription factors play key roles in determining neuronal subtype identity in flies and vertebrates. dbx belongs to the highly divergent H2.0 family of homeobox genes. In vertebrates, Dbx1 and Dbx2 promote the development of a subset of interneurons, some of which help mediate left-right coordination of locomotor activity. Here, we identify and show that the single Drosophila ortholog of Dbx1/2 contributes to the development of specific subsets of interneurons via cross-repressive, lineage-specific interactions with the motoneuron-promoting factors eve and hb9 (exex). dbx is expressed primarily in interneurons of the embryonic, larval and adult central nervous system, and these interneurons tend to extend short axons and be GABAergic. Interestingly, many Dbx(+) interneurons share a sibling relationship with Eve(+) or Hb9(+) motoneurons. The non-overlapping expression of dbx and eve, or dbx and hb9, within pairs of sibling neurons is initially established as a result of Notch/Numb-mediated asymmetric divisions. Cross-repressive interactions between dbx and eve, and dbx and hb9, then help maintain the distinct expression profiles of these genes in their respective pairs of sibling neurons. Strict maintenance of the mutually exclusive expression of dbx relative to that of eve and hb9 in sibling neurons is crucial for proper neuronal specification, as misexpression of dbx in motoneurons dramatically hinders motor axon outgrowth.

  18. 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

  19. Repression of Rgg But Not Upregulation of LacD.1 in emm1-type covS Mutant Mediates the SpeB Repression in Group A Streptococcus

    PubMed Central

    Chiang-Ni, Chuan; Chu, Teng-Ping; Wu, Jiunn-Jong; Chiu, Cheng-Hsun

    2016-01-01

    CovR/CovS is an important two-component regulatory system in human pathogen group A Streptococcus (GAS). Epidemiological studies have shown that inactivation of the sensor kinase CovS is correlated with invasive clinical manifestations. The phosphorylation level of response regulator CovR decreases dramatically in the absence of CovS, resulting in the derepression of virulence factor expression and an increase in bacterial invasiveness. Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease and is negatively regulated by CovR; however, the expression of SpeB is almost completely repressed in the covS mutant. The present study found that in the emm1-type A20 strain, non-phosphorylated CovR acts as a transcriptional repressor for SpeB-positive regulator Rgg. In addition, the expression of Rgg-negative regulator LacD.1 is upregulated in the covS mutant. These results suggest that inactivation of Rgg in the covS mutant would directly mediate speB repression. The current study showed that overexpression of rgg but not inactivation of lacD.1 in the covS mutant partially restores speB expression, indicating that only rgg repression, but not lacD.1 upregulation, contributes to the speB repression in the covS mutant. PMID:27965655

  20. Chromatin Accessibility Mapping Identifies Mediators of Basal Transcription and Retinoid-Induced Repression of OTX2 in Medulloblastoma

    PubMed Central

    Zhang, Monica; Song, Lingyun; Lee, Bum-Kyu; Iyer, Vishwanath R.; Furey, Terrence S.; Crawford, Gregory E.; Yan, Hai; He, Yiping

    2014-01-01

    Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes. PMID:25198066

  1. 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

  2. miR-25/93 mediates hypoxia-induced immunosuppression by repressing cGAS.

    PubMed

    Wu, Min-Zu; Cheng, Wei-Chung; Chen, Su-Feng; Nieh, Shin; O'Connor, Carolyn; Liu, Chia-Lin; Tsai, Wen-Wei; Wu, Cheng-Jang; Martin, Lorena; Lin, Yaoh-Shiang; Wu, Kou-Juey; Lu, Li-Fan; Izpisua Belmonte, Juan Carlos

    2017-10-01

    The mechanisms by which hypoxic tumours evade immunological pressure and anti-tumour immunity remain elusive. Here, we report that two hypoxia-responsive microRNAs, miR-25 and miR-93, are important for establishing an immunosuppressive tumour microenvironment by downregulating expression of the DNA sensor cGAS. Mechanistically, miR-25/93 targets NCOA3, an epigenetic factor that maintains basal levels of cGAS expression, leading to repression of cGAS during hypoxia. This allows hypoxic tumour cells to escape immunological responses induced by damage-associated molecular pattern molecules, specifically the release of mitochondrial DNA. Moreover, restoring cGAS expression results in an anti-tumour immune response. Clinically, decreased levels of cGAS are associated with poor prognosis for patients with breast cancer harbouring high levels of miR-25/93. Together, these data suggest that inactivation of the cGAS pathway plays a critical role in tumour progression, and reveal a direct link between hypoxia-responsive miRNAs and adaptive immune responses to the hypoxic tumour microenvironment, thus unveiling potential new therapeutic strategies.

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

    PubMed

    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

    2016-07-29

    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.

  4. HDAC dependent transcriptional repression of Bmp-7 potentiates TGF-β mediated renal fibrosis in obstructive uropathy.

    PubMed

    Manson, Scott R; Song, Joseph B; Hruska, Keith A; Austin, Paul F

    2014-01-01

    Recombinant BMP-7 inhibits the pathogenesis of renal injury in response to various stimuli. However, little is known about the molecular regulation of endogenous BMP-7 and its renal protective functions. We examined transcriptional regulation of Bmp-7 and its role in the pathogenesis of renal injury resulting from urinary tract dysfunction. Obstruction induced renal injury was modeled in vivo in mice by unilateral ureteral obstruction and in vitro in primary kidney cells by treatment with transforming growth factor-β, a profibrotic cytokine that is increased in the obstructed kidney. Unilateral ureteral obstruction resulted in the loss of BMP-7 expression in conjunction with histone deacetylation and transcriptional repression of the Bmp-7 promoter. The histone deacetylase inhibitor trichostatin A stimulated Bmp-7 expression in primary kidney cells. Trichostatin A also inhibited the expression of transforming growth factor-β dependent profibrotic genes in a manner that depended on BMP receptor signaling. These findings extended to the obstructed kidney in vivo, in which trichostatin A treatment restored the expression of Bmp-7 along with BMP-7 mediated suppression of transforming growth factor-β dependent signaling pathways. Finally, trichostatin A stimulated activation of the BMP-7 pathway the ameliorated obstruction induced renal injury by preventing disruption of the renal architecture and the development of renal fibrosis. These findings show that histone deacetylase dependent repression of Bmp-7 transcription is a critical event during the pathogenesis of renal injury in obstructive uropathy. Accordingly, treatment with histone deacetylase inhibitors represents a potentially effective strategy to restore BMP-7 expression and its renal protective functions during treatment of obstructive uropathy. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  5. 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

  6. Glucocorticoid Receptor Signaling Represses the Antioxidant Response by Inhibiting Histone Acetylation Mediated by the Transcriptional Activator NRF2.

    PubMed

    Alam, Md Morshedul; Okazaki, Keito; Nguyen, Linh Thi Thao; Ota, Nao; Kitamura, Hiroshi; Murakami, Shohei; Shima, Hiroki; Igarashi, Kazuhiko; Sekine, Hiroki; Motohashi, Hozumi

    2017-03-17

    NRF2 (nuclear factor erythroid 2-related factor 2) is a key transcriptional activator that mediates the inducible expression of antioxidant genes. NRF2 is normally ubiquitinated by KEAP1 (Kelch-like ECH-associated protein 1) and subsequently degraded by proteasomes. Inactivation of KEAP1 by oxidative stress or electrophilic chemicals allows NRF2 to activate transcription through binding to antioxidant response elements (AREs) and recruiting histone acetyltransferase CBP (CREB-binding protein). While KEAP1-dependent regulation is a major determinant of NRF2 activity, NRF2-mediated transcriptional activation varies from context to context, suggesting other intracellular signaling cascades may impact NRF2 function. To identify a signaling pathway that modifies NRF2 activity, we immunoprecipitated endogenous NRF2 and its interacting proteins from mouse liver and identified glucocorticoid receptor (GR) as a novel NRF2-binding partner. We found that glucocorticoids (GC), dexamethasone (Dex) and betamethasone (Bet), antagonize diethyl maleate (DEM)-induced activation of NRF2 target genes in a GR-dependent manner. Dex treatment enhanced GR recruitment to AREs without affecting chromatin binding of NRF2, resulting in the inhibition of CBP recruitment and histone acetylation at AREs. This repressive effect was canceled by the addition of HDAC inhibitors. Thus, GR signaling decreases NRF2 transcriptional activation through reducing the NRF2-dependent histone acetylation. Consistent with these observations, GR signaling blocked NRF2-mediated cytoprotection from oxidative stress. This study suggests that an impaired antioxidant response by NRF2 and a resulting decrease in cellular antioxidant capacity account for the side effects of GCs, providing a novel viewpoint for the pathogenesis of hypercorticosteroidism.

  7. 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.

  8. 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.

  9. S-phase-dependent action of cycloheximide in relieving chromatin-mediated general transcriptional repression.

    PubMed Central

    Cesari, M; Héliot, L; Meplan, C; Pabion, M; Khochbin, S

    1998-01-01

    Chromatin plays a major role in the tight regulation of gene expression and in constraining inappropriate gene activity. Replication-coupled chromatin assembly ensures maintenance of these functions of chromatin during S phase of the cell cycle. Thus treatment of cells with an inhibitor of translation, such as cycloheximide (CX), would be expected to have a dramatic effect on chromatin structure and function, essentially in S phase of the cell cycle, due to uncoupled DNA replication and chromatin assembly. In this work, we confirm this hypothesis and show that CX can induce a dramatic S-phase-dependent alteration in chromatin structure that is associated with general RNA polymerase II-dependent transcriptional activation. Using two specific RNA polymerase II-transcribed genes, we confirm the above conclusion and show that CX-mediated transcriptional activation is enhanced during the DNA replication phase of the cell cycle. Moreover, we show co-operation between an inhibitor of histone deacetylase and CX in inducing gene expression, which is again S-phase-dependent. The modest effect of CX in inducing the activity of a transiently transfected promoter shows that the presence of the promoter in an endogenous chromatin context is necessary in order to observe transcriptional activation. We therefore suggest that the uncoupled DNA replication and histone synthesis that occur after CX treatment induces a general modification of chromatin structure, and propose that this general disorganization of chromatin structure is responsible for a widespread activation of RNA polymerase II-mediated gene transcription. PMID:9841873

  10. Two Distinct Mechanisms Govern RpoS-Mediated Repression of Tick-Phase Genes during Mammalian Host Adaptation by Borrelia burgdorferi, the Lyme Disease Spirochete

    PubMed Central

    Grove, Arianna P.; Liveris, Dionysios; Iyer, Radha; Petzke, Mary; Rudman, Joseph; Caimano, Melissa J.; Radolf, Justin D.

    2017-01-01

    ABSTRACT The alternative sigma factor RpoS plays a key role modulating gene expression in Borrelia burgdorferi, the Lyme disease spirochete, by transcribing mammalian host-phase genes and repressing σ70-dependent genes required within the arthropod vector. To identify cis regulatory elements involved in RpoS-dependent repression, we analyzed green fluorescent protein (GFP) transcriptional reporters containing portions of the upstream regions of the prototypical tick-phase genes ospAB, the glp operon, and bba74. As RpoS-mediated repression occurs only following mammalian host adaptation, strains containing the reporters were grown in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats. Wild-type spirochetes harboring ospAB- and glp-gfp constructs containing only the minimal (−35/−10) σ70 promoter elements had significantly lower expression in DMCs relative to growth in vitro at 37°C; no reduction in expression occurred in a DMC-cultivated RpoS mutant harboring these constructs. In contrast, RpoS-mediated repression of bba74 required a stretch of DNA located between −165 and −82 relative to its transcriptional start site. Electrophoretic mobility shift assays employing extracts of DMC-cultivated B. burgdorferi produced a gel shift, whereas extracts from RpoS mutant spirochetes did not. Collectively, these data demonstrate that RpoS-mediated repression of tick-phase borrelial genes occurs by at least two distinct mechanisms. One (e.g., ospAB and the glp operon) involves primarily sequence elements near the core promoter, while the other (e.g., bba74) involves an RpoS-induced transacting repressor. Our results provide a genetic framework for further dissection of the essential “gatekeeper” role of RpoS throughout the B. burgdorferi enzootic cycle. PMID:28830947

  11. The crystal structure of the AhRR/ARNT heterodimer reveals the structural basis of the repression of AhR-mediated transcription.

    PubMed

    Sakurai, Shunya; Shimizu, Toshiyuki; Ohto, Umeharu

    2017-09-13

    2,3,7,8-Tetrachlorodibenzo-p-dioxin and related compounds (TCDDs) are extraordinarily potent environmental toxic pollutants. Most of the TCDD toxicities are mediated by aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor belonging to the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) family. Upon ligand binding, AhR forms a heterodimer with AhR nuclear translocator (ARNT) and induces the expression of genes involved in various biological responses. One of the genes induced by AhR encodes AhR repressor (AhRR), which also forms a heterodimer with ARNT and represses the activation of AhR-dependent transcription. The control of AhR activation is critical for managing AhR-mediated diseases, but the mechanisms by which AhRR represses AhR activation remain poorly understood, due to the lack of structural information. Here, we determined the structure of the AhRR/ARNT heterodimer by X-ray crystallography, which revealed an asymmetric intertwined domain organization presenting structural features that are both conserved and distinct among bHLH-PAS family members. The structures of AhRR/ARNT and AhR/ARNT were similar in the bHLH-PAS-A region, while the PAS-B of ARNT in the AhRR/ARNT complex exhibited a different domain arrangement in this family reported so far. The structure clearly disclosed that AhRR competitively represses AhR binding to ARNT and target DNA, and further suggested the existence of an AhRR/ARNT-specific repression mechanism. This study provides a structural basis for understanding the mechanism by which AhRR represses AhR-mediated gene transcription. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  12. 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.

  13. The Pluripotency Factor NANOG Binds to GLI Proteins and Represses Hedgehog-mediated Transcription*

    PubMed Central

    Li, Qiang; Lex, Rachel K.; Chung, HaeWon; Giovanetti, Simone M.; Ji, Zhicheng; Ji, Hongkai; Person, Maria D.; Kim, Jonghwan; Vokes, Steven A.

    2016-01-01

    The Hedgehog (HH) signaling pathway is essential for the maintenance and response of several types of stem cells. To study the transcriptional response of stem cells to HH signaling, we searched for proteins binding to GLI proteins, the transcriptional effectors of the HH pathway in mouse embryonic stem (ES) cells. We found that both GLI3 and GLI1 bind to the pluripotency factor NANOG. The ectopic expression of NANOG inhibits GLI1-mediated transcriptional responses in a dose-dependent fashion. In differentiating ES cells, the presence of NANOG reduces the transcriptional response of cells to HH. Finally, we found that Gli1 and Nanog are co-expressed in ES cells at high levels. We propose that NANOG acts as a negative feedback component that provides stem cell-specific regulation of the HH pathway. PMID:26797124

  14. TBP-like protein (TLP) interferes with Taspase1-mediated processing of TFIIA and represses TATA box gene expression.

    PubMed

    Suzuki, Hidefumi; Isogai, Momoko; Maeda, Ryo; Ura, Kiyoe; Tamura, Taka-Aki

    2015-07-27

    TBP-TFIIA interaction is involved in the potentiation of TATA box-driven promoters. TFIIA activates transcription through stabilization of TATA box-bound TBP. The precursor of TFIIA is subjected to Taspase1-directed processing to generate α and β subunits. Although this processing has been assumed to be required for the promoter activation function of TFIIA, little is known about how the processing is regulated. In this study, we found that TBP-like protein (TLP), which has the highest affinity to TFIIA among known proteins, affects Taspase1-driven processing of TFIIA. TLP interfered with TFIIA processing in vivo and in vitro, and direct binding of TLP to TFIIA was essential for inhibition of the processing. We also showed that TATA box promoters are specifically potentiated by processed TFIIA. Processed TFIIA, but not unprocessed TFIIA, associated with the TATA box. In a TLP-knocked-down condition, not only the amounts of TATA box-bound TFIIA but also those of chromatin-bound TBP were significantly increased, resulting in the stimulation of TATA box-mediated gene expression. Consequently, we suggest that TLP works as a negative regulator of the TFIIA processing and represses TFIIA-governed and TATA-dependent gene expression through preventing TFIIA maturation.

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

    PubMed

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

    2016-01-04

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

  16. pVHL-Mediated Transcriptional Repression of c-Myc by Recruitment of Histone Deacetylases

    PubMed Central

    Hwang, In-Young; Roe, Jae-Seok; Seol, Ja-Hwan; Kim, Hwa-Ryeon; Cho, Eun-Jung; Youn, Hong-Duk

    2012-01-01

    The biological functions of Myc are to regulate cell growth, apoptosis, cell differentiation and stem-cell self-renewal. Abnormal accumulation of c-Myc is able to induce excessive proliferation of normal cells. von Hippel-Lindau protein (pVHL) is a key regulator of hypoxia-inducible factor1α (HIF1α), thus accumulation and hyperactivation of HIF1α is the most prominent feature of VHL-mutated renal cell carcinoma. Interestingly, the Myc pathway is reported to be activated in renal cell carcinoma even though the precise molecular mechanism still remains to be established. Here, we demonstrated that pVHL locates at the c-Myc promoter region through physical interaction with Myc. Furthermore, pVHL reinforces HDAC1/2 recruitment to the Myc promoter, which leads to the auto-suppression of Myc. Therefore, one possible mechanism of Myc auto-suppression by pVHL entails removing histone acetylation. Our study identifies a novel mechanism for pVHL-mediated negative regulation of c-Myc transcription. PMID:22286234

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

    PubMed Central

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

    2015-01-01

    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

  18. Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors

    PubMed Central

    Levanon, Ditsa; Goldstein, Robert E.; Bernstein, Yael; Tang, Hua; Goldenberg, Dalia; Stifani, Stefano; Paroush, Ze’ev; Groner, Yoram

    1998-01-01

    The mammalian AML/CBFα runt domain (RD) transcription factors regulate hematopoiesis and osteoblast differentiation. Like their Drosophila counterparts, most mammalian RD proteins terminate in a common pentapeptide, VWRPY, which serves to recruit the corepressor Groucho (Gro). Using a yeast two-hybrid assay, in vitro association and pull-down experiments, we demonstrate that Gro and its mammalian homolog TLE1 specifically interact with AML1 and AML2. In addition to the VWRPY motif, other C-terminal sequences are required for these interactions with Gro/TLE1. TLE1 inhibits AML1-dependent transactivation of the T cell receptor (TCR) enhancers α and β, which contain functional AML binding sites, in transfected Jurkat T cells. LEF-1 is an additional transcription factor that mediates transactivation of TCR enhancers. LEF-1 and its Drosophila homolog Pangolin (Pan) are involved in the Wnt/Wg signaling pathway through interactions with the coactivator β-catenin and its highly conserved fly homolog Armadillo (Arm). We show that TLE/Gro interacts with LEF-1 and Pan, and inhibits LEF-1:β-catenin-dependent transcription. These data indicate that, in addition to their activity as transcriptional activators, AML1 and LEF-1 can act, through recruitment of the corepressor TLE1, as transcriptional repressors in TCR regulation and Wnt/Wg signaling. PMID:9751710

  19. 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.

  20. Regulation of miRNA Processing and miRNA Mediated Gene Repression in Cancer

    PubMed Central

    Bajan, Sarah; Hutvagner, Gyorgy

    2014-01-01

    The majority of human protein-coding genes are predicted to be targets of miRNA-mediated post-transcriptional regulation. The widespread influence of miRNAs is illustrated by their essential roles in all biological processes. Regulated miRNA expression is essential for maintaining cellular differentiation; therefore alterations in miRNA expression patterns are associated with several diseases, including various cancers. High-throughput sequencing technologies revealed low level expressing miRNA isoforms, termed isomiRs. IsomiRs may differ in sequence, length, target preference and expression patterns from their parental miRNA and can arise from differences in miRNA biosynthesis, RNA editing, or SNPs inherent to the miRNA gene. The association between isomiR expression and disease progression is largely unknown. Misregulated miRNA expression is thought to contribute to the formation and/or progression of cancer. However, due to the diversity of targeted transcripts, miRNAs can function as both tumor-suppressor genes and oncogenes as defined by cellular context. Despite this, miRNA profiling studies concluded that the differential expression of particular miRNAs in diseased tissue could aid the diagnosis and treatment of some cancers. PMID:25069508

  1. Complementary quantitative proteomics reveals that transcription factor AP-4 mediates E-box-dependent complex formation for transcriptional repression of HDM2.

    PubMed

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

    2009-09-01

    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 a

  2. 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

  3. Calcium-mediated repression of β-catenin and its transcriptional signaling mediates neural crest cell death in an avian model of fetal alcohol syndrome.

    PubMed

    Flentke, George R; Garic, Ana; Amberger, Ed; Hernandez, Marcos; Smith, Susan M

    2011-07-01

    Fetal alcohol syndrome (FAS) is a common birth defect in many societies. Affected individuals have neurodevelopmental disabilities and a distinctive craniofacial dysmorphology. These latter deficits originate during early development from the ethanol-mediated apoptotic depletion of cranial facial progenitors, a population known as the neural crest. We showed previously that this apoptosis is caused because acute ethanol exposure activates G-protein-dependent intracellular calcium within cranial neural crest progenitors, and this calcium transient initiates the cell death. The dysregulated signals that reside downstream of ethanol's calcium transient and effect neural crest death are unknown. Here we show that ethanol's repression of the transcriptional effector β-catenin causes the neural crest losses. Clinically relevant ethanol concentrations (22-78 mM) rapidly deplete nuclear β-catenin from neural crest progenitors, with accompanying losses of β-catenin transcriptional activity and downstream genes that govern neural crest induction, expansion, and survival. Using forced expression studies, we show that β-catenin loss of function (via dominant-negative T cell transcription factor [TCF]) recapitulates ethanol's effects on neural crest apoptosis, whereas β-catenin gain-of-function in ethanol's presence preserves neural crest survival. Blockade of ethanol's calcium transient using Bapta-AM normalizes β-catenin activity and prevents the neural crest losses, whereas ionomycin treatment is sufficient to destabilize β-catenin. We propose that ethanol's repression of β-catenin causes the neural crest losses in this model of FAS. β-Catenin is a novel target for ethanol's teratogenicity. β-Catenin/Wnt signals participate in many developmental events and its rapid and persistent dysregulation by ethanol may explain why the latter is such a potent teratogen.

  4. Retinoid X receptor alpha represses GATA-4-mediated transcription via a retinoid-dependent interaction with the cardiac-enriched repressor FOG-2.

    PubMed

    Clabby, Martha L; Robison, Trevor A; Quigley, Heather F; Wilson, David B; Kelly, Daniel P

    2003-02-21

    Dietary vitamin A and its derivatives, retinoids, regulate cardiac growth and development. To delineate mechanisms involved in retinoid-mediated control of cardiac gene expression, the regulatory effects of the retinoid X receptor alpha (RXR alpha) on atrial naturietic factor (ANF) gene transcription was investigated. The transcriptional activity of an ANF promoter-reporter in rat neonatal ventricular myocytes was repressed by RXR alpha in the presence of 9-cis-RA and by the constitutively active mutant RXR alpha F318A indicating that liganded RXR confers the regulatory effect. The RXR alpha-mediated repression mapped to the proximal 147 bp of the rat ANF promoter, a region lacking a consensus retinoid response element but containing several known cardiogenic cis elements including a well characterized GATA response element. Glutathione S-transferase "pull-down" assays revealed that RXR alpha interacts directly with GATA-4, in a ligand-independent manner, via the DNA binding domain of RXR alpha and the second zinc finger of GATA-4. Liganded RXR alpha repressed the activity of a heterologous promoter-reporter construct containing GATA-response element recognition sites in cardiac myocytes but not in several other cell types, suggesting that additional cardiac-enriched factors participate in the repression complex. Co-transfection of liganded RXR alpha and the known cardiac-enriched GATA-4 repressor, FOG-2, resulted in additive repression of GATA-4 activity in ventricular myocytes. In addition, RXR alpha was found to bind FOG-2, in a 9-cis-RA-dependent manner. These data reveal a novel mechanism by which retinoids regulate cardiogenic gene expression through direct interaction with GATA-4 and its co-repressor, FOG-2.

  5. Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis

    PubMed Central

    Yang, Li; Wu, Gang; Poethig, R. Scott

    2012-01-01

    Plant microRNAs (miRNAs) typically mediate RNA cleavage, but examples of miRNA-mediated translational repression have also been reported. However, the functional significance of this latter process is unknown. We identified SUO in a screen for Arabidopsis mutations that increase the accumulation of the miR156-regulated gene SPL3. suo has a loss-of-function phenotype characteristic of plants with reduced Argonaute (AGO)1 activity. An analysis of RNA and protein levels in suo mutants demonstrated that this phenotype is a consequence of a defect in miRNA-mediated translational repression; the effect of suo on vegetative phase change is attributable to a reduction in miR156/miR157 activity. SUO encodes a large protein with N-terminal bromo-adjacent homology (BAH) and transcription elongation factor S-II (TFS2N) domains and two C-terminal GW (glycine and tryptophan) repeats. SUO is present in the nucleus, and colocalizes with the processing-body component DCP1 in the cytoplasm. Our results reveal that SOU is a component of the miRNA pathway in Arabidopsis and demonstrate that translational repression is a functionally important aspect of miRNA activity in plants. PMID:22184231

  6. Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis.

    PubMed

    Yang, Li; Wu, Gang; Poethig, R Scott

    2012-01-03

    Plant microRNAs (miRNAs) typically mediate RNA cleavage, but examples of miRNA-mediated translational repression have also been reported. However, the functional significance of this latter process is unknown. We identified SUO in a screen for Arabidopsis mutations that increase the accumulation of the miR156-regulated gene SPL3. suo has a loss-of-function phenotype characteristic of plants with reduced Argonaute (AGO)1 activity. An analysis of RNA and protein levels in suo mutants demonstrated that this phenotype is a consequence of a defect in miRNA-mediated translational repression; the effect of suo on vegetative phase change is attributable to a reduction in miR156/miR157 activity. SUO encodes a large protein with N-terminal bromo-adjacent homology (BAH) and transcription elongation factor S-II (TFS2N) domains and two C-terminal GW (glycine and tryptophan) repeats. SUO is present in the nucleus, and colocalizes with the processing-body component DCP1 in the cytoplasm. Our results reveal that SOU is a component of the miRNA pathway in Arabidopsis and demonstrate that translational repression is a functionally important aspect of miRNA activity in plants.

  7. 15-Deoxy-Δ12,14-prostaglandin J2 induces expression of 15-hydroxyprostaglandin dehydrogenase through Elk-1 activation in human breast cancer MDA-MB-231 cells.

    PubMed

    Kim, Hye-Rim; Lee, Ha-Na; Lim, Kyu; Surh, Young-Joon; Na, Hye-Kyung

    2014-10-01

    Overproduction of prostaglandin E2 (PGE2) has been reported to be implicated in carcinogenesis. The intracellular level of PGE2 is maintained not only by its biosynthesis, but also by inactivation/degradation. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme that catalyzes the conversion of oncogenic PGE2 to a biologically inactive keto metabolite. In the present study, we demonstrate that 15-deoxy-Δ(12,14)-prostaglandin J2 (15 d-PGJ2), one of the terminal products of cyclooxygenase-2, updregulates the expression and the activity of 15-PGDH in human breast cancer MDA-MB-231 cells. By using deletion constructs of the 15-PGDH promoter, we have found that E-twenty six (Ets) is the most essential determinant for 15-PGDH induction. 15 d-PGJ2 induced phosphorylation of Elk-1, one of Ets transcription factor family members, in the nucleus. Knockdown of Elk-1 abolished the ability of 15 d-PGJ2 to upregulate 15-PGDH expression. Furthermore, 15 d-PGJ2-mediated activation of Elk-1 was found to be dependent on activation of extracellular-signal related kinase (ERK) 1/2. Treatment of U0126, a pharmacological inhibitor of MEK1/2-ERK, abolished phosphorylation and DNA binding of Elk-1 as well as 15-PGDH induction in 15 d-PGJ2-treated MDA-MB-231 cells. Moreover, 15 d-PGJ2 generated reactive oxygen species (ROS), which contribute to the expression of 15-PGDH as well as phosphorylation of ERK1/2 and Elk-1. 15 d-PGJ2 inhibited the migration of MDA-MB-231 cells, which was attenuated by transient transfection with 15-PGDH siRNA. Taken together, these findings suggest that 15 d-PGJ2 induces the expression of 15-PGDH through ROS-mediated activation of ERK1/2 and subsequently Elk-1 in the MDA-MB-231 cells, which may contribute to tumor suppressive activity of this cyclopentenone prostaglandin.

  8. Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins[S

    PubMed Central

    Subra, Caroline; Grand, David; Laulagnier, Karine; Stella, Alexandre; Lambeau, Gérard; Paillasse, Michael; De Medina, Philippe; Monsarrat, Bernard; Perret, Bertrand; Silvente-Poirot, Sandrine; Poirot, Marc; Record, Michel

    2010-01-01

    Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA2-IVA, the calcium-independent iPLA2-VIA, and the secreted sPLA2-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPγS triggered activation of phospholipase A2 (PLA2)and PLD2. A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandinJ2 (15-d PGJ2), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell. PMID:20424270

  9. 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.

  10. 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.

  11. 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.

  12. 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. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  13. 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

  14. Xenopus CAF1 requires NOT1-mediated interaction with 4E-T to repress translation in vivo.

    PubMed

    Waghray, Shruti; Williams, Clay; Coon, Joshua J; Wickens, Marvin

    2015-07-01

    RNA-regulatory factors bound to 3' UTRs control translation and stability. Repression often is associated with poly(A) removal. The deadenylase CAF1 is a core component of the CCR4-NOT complex. Our prior studies established that CAF1 represses translation independent of deadenylation. We sought the mechanism of its deadenylation-independent repression in Xenopus oocytes. Our data reveal a chain of interacting proteins that links CAF1 to CCR4-NOT and to Xp54 and 4E-T. Association of CAF1 with NOT1, the major subunit of CCR4-NOT, is required for repression by CAF1 tethered to a reporter mRNA. Affinity purification-mass spectrometry and coimmunoprecipitation revealed that at least five members of the CCR4-NOT complex were recruited by CAF1. The recruitment of these proteins required NOT1, as did the ability of tethered CAF1 to repress translation. In turn, NOT1 was needed to recruit Xp54 and 4E-T. We examined the role of 4E-T in repression using mutations that disrupted either eIF4E-dependent or -independent mechanisms. Expression of a 4E-T truncation that still bound eIF4E alleviated repression by tethered CAF1, NOT1, and Xp54. In contrast, a mutant 4E-T that failed to bind eIF4E did not. Repression of global translation was affected only by the eIF4E-dependent mechanism. Reporters bearing IRES elements revealed that repression via tethered CAF1 and Xp54 is cap- and eIF4E-independent, but requires one or more of eIF4A, eIF4B, and eIF4G. We propose that RNA-binding proteins, and perhaps miRNAs, repress translation through an analogous chain of interactions that begin with the 3' UTR-bound repressor and end with the noncanonical activity of 4E-T.

  15. 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.

  16. Major histocompatibility class I gene transcription in thyrocytes: a series of interacting regulatory DNA sequence elements mediate thyrotropin/cyclic adenosine 3',5'-monophosphate repression.

    PubMed

    Kirshner, S; Palmer, L; Bodor, J; Saji, M; Kohn, L D; Singer, D S

    2000-01-01

    In response to TSH, thyroid cells decrease major histocompatibility (MHC) class I expression and transcription, providing an excellent model for studying the dynamic modulation of transcription of MHC class I genes. Here we show that protein kinase A (PKA), a downstream effector of the TSH/cAMP pathway, reproduces the effects of TSH in repressing class I transcription. PKA/cAMP-mediated repression of transcription involves multiple interacting upstream response elements in the class I promoter: an element extending from -127 to -90 bp containing a CRE-like core, and at least two elements within an upstream 30-bp segment (-160 to -130 bp), which overlaps with the interferon regulatory element. ICER (inducible cAMP early response), a transcriptional repressor induced by TSH/cAMP can decrease class I promoter activity when introduced into FRTL-5 thyroid cells in the absence of TSH/cAMP. ICER binds to both the CRE-like element and the upstream 30-bp segment, generating a novel TSH-induced ternary complex. The present studies led to the proposal that TSH-mediated repression of class I transcription is the result of integrating signals from transcription factors through the higher order interactions of multiple regulatory elements.

  17. Growth Suppression by Acute Promyelocytic Leukemia-Associated Protein PLZF Is Mediated by Repression of c-myc Expression

    PubMed Central

    McConnell, Melanie J.; Chevallier, Nathalie; Berkofsky-Fessler, Windy; Giltnane, Jena M.; Malani, Rupal B.; Staudt, Louis M.; Licht, Jonathan D.

    2003-01-01

    The transcriptional repressor PLZF was identified by its translocation with retinoic acid receptor alpha in t(11;17) acute promyelocytic leukemia (APL). Ectopic expression of PLZF leads to cell cycle arrest and growth suppression, while disruption of normal PLZF function is implicated in the development of APL. To clarify the function of PLZF in cell growth and survival, we used an inducible PLZF cell line in a microarray analysis to identify the target genes repressed by PLZF. One prominent gene identified was c-myc. The array analysis demonstrated that repression of c-myc by PLZF led to a reduction in c-myc-activated transcripts and an increase in c-myc-repressed transcripts. Regulation of c-myc by PLZF was shown to be both direct and reversible. An interaction between PLZF and the c-myc promoter could be detected both in vitro and in vivo. PLZF repressed the wild-type c-myc promoter in a reporter assay, dependent on the integrity of the binding site identified in vitro. PLZF binding in vivo was coincident with a decrease in RNA polymerase occupation of the c-myc promoter, indicating that repression occurred via a reduction in the initiation of transcription. Finally, expression of c-myc reversed the cell cycle arrest induced by PLZF. These data suggest that PLZF expression maintains a cell in a quiescent state by repressing c-myc expression and preventing cell cycle progression. Loss of this repression through the translocation that occurs in t(11;17) would have serious consequences for cell growth control. PMID:14645547

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

    PubMed

    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

    2010-01-20

    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.

  19. Glucocorticoid-Mediated Repression of the Oncogenic microRNA Cluster miR-17∼92 Contributes to the Induction of Bim and Initiation of Apoptosis

    PubMed Central

    Molitoris, Jason K.; McColl, Karen S.

    2011-01-01

    Synthetic glucocorticoids were one of the first effective treatments for lymphoid malignancies because of their ability to induce apoptosis and are still used in combination with other chemotherapeutic agents. Up-regulation of Bim, a proapoptotic member of the B-cell lymphoma-2 family, is an important mediator of glucocorticoid-induced apoptosis. Although glucocorticoids are known to elevate Bim mRNA and protein, little is known about the mechanism. Here, we report that glucocorticoids repress the expression of the microRNA cluster miR-17∼92, which results in elevated Bim protein expression as a mechanism by which glucocorticoids induce Bim. Using a luciferase-Bim 3′ untranslated region construct, we demonstrate that glucocorticoids mediate Bim induction posttranscriptionally after miR-17∼92 repression, resulting in increased Bim protein expression. Overexpression of miR-17∼92 microRNAs decreases Bim induction and attenuates glucocorticoid-mediated apoptosis. Conversely, knockdown of miR-17∼92 increases Bim protein expression and glucocorticoid-mediated apoptosis. These findings indicate that endogenous levels of miR-17∼92 repress Bim expression in T-cell lymphoid malignancies and that glucocorticoids induce Bim expression via down-regulation of the miR-17∼92 microRNA cluster. Our findings present a novel mechanism that contributes to the up-regulation of Bim and induction of apoptosis in lymphocytes after glucocorticoid treatment. Furthermore, our work demonstrating that inhibition of miR-17∼92 increases glucocorticoid-induced apoptosis highlights the potential importance of miR-17∼92 as a therapeutic target in leukemias and lymphomas. PMID:21239610

  20. HPrK Regulates Succinate-Mediated Catabolite Repression in the Gram-Negative Symbiont Sinorhizobium meliloti▿

    PubMed Central

    Pinedo, Catalina Arango; Gage, Daniel J.

    2009-01-01

    The HPrK kinase/phosphatase is a common component of the phosphotransferase system (PTS) of gram-positive bacteria and regulates catabolite repression through phosphorylation/dephosphorylation of its substrate, the PTS protein HPr, at a conserved serine residue. Phosphorylation of HPr by HPrK also affects additional phosphorylation of HPr by the PTS enzyme EI at a conserved histidine residue. Sinorhizobium meliloti can live as symbionts inside legume root nodules or as free-living organisms and is one of the relatively rare gram-negative bacteria known to have a gene encoding HPrK. We have constructed S. meliloti mutants that lack HPrK or that lack key amino acids in HPr that are likely phosphorylated by HPrK and EI. Deletion of hprK in S. meliloti enhanced catabolite repression caused by succinate, as did an S53A substitution in HPr. Introduction of an H22A substitution into HPr alleviated the strong catabolite repression phenotypes of strains carrying ΔhprK or hpr(S53A) mutations, demonstrating that HPr-His22-P is needed for strong catabolite repression. Furthermore, strains with a hpr(H22A) allele exhibited relaxed catabolite repression. These results suggest that HPrK phosphorylates HPr at the serine-53 residue, that HPr-Ser53-P inhibits phosphorylation at the histidine-22 residue, and that HPr-His22-P enhances catabolite repression in the presence of succinate. Additional experiments show that ΔhprK mutants overproduce exopolysaccharides and form nodules that do not fix nitrogen. PMID:18931135

  1. Activation of PPARγ does not contribute to macrophage ABCA1 expression and ABCA1-mediated cholesterol efflux to apoAI.

    PubMed

    Jiang, Meixiu; Li, Xiaoju

    2017-01-22

    Activation of macrophage ABCA1/G1 expression and cholesterol efflux is believed one of the mechanisms by which PPARγ inhibits atherosclerosis. PPARγ can also activate CD36 expression, a receptor for oxLDL, which may supply LXR ligands to activate LXR-ABCA1/G1 pathways. However, the controversial effects of PPARγ on ABCA1 expression have been reported. In this study, we used peritoneal macrophages isolated from wild type and CD36 deficient (CD36(-/-)) mice to clarify if PPARγ ligands can influence ABCA1 expression by CD36 function. We found that CD36 deficiency had no effect on cholesterol efflux and ABCA1/ABCG1 expression at basal levels. In both cell types, PPARγ ligands (15d-PGJ2, troglitazone and pioglitazone) reduced ABCA1 expression and ABCA1-mediated cholesterol efflux to apoAI, with most by troglitazone. LXR ligand-induced ABCA1 expression and cholesterol efflux was attenuated by PPARγ ligands. Associated with decreased ABCA1 protein levels, ABCA1 mRNA and promoter activity were reduced by PPARγ ligands. Furthermore, high expressing PPARγ reduced ABCA1 expression and LXR-activated ABCA1 promoter in a CD36-independent manner. In contrast, ABCG1 expression was induced by PPARγ ligands while inhibited by PPARγ inactivation. Taken together, our study suggests that enhancement of macrophage cholesterol metabolism by PPARγ is not contributed by activating ABCA1 expression and ABCA1-mediated cholesterol efflux to apoAI, which is not involved by CD36 expression either. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Repression of oxalic acid-mediated mineral phosphate solubilization in rhizospheric isolates of Klebsiella pneumoniae by succinate.

    PubMed

    Rajput, Mahendrapal Singh; Naresh Kumar, G; Rajkumar, Shalini

    2013-02-01

    Two strains of Klebsiella (SM6 and SM11) were isolated from rhizospheric soil that solubilized mineral phosphate by secretion of oxalic acid from glucose. Activities of enzymes for periplasmic glucose oxidation (glucose dehydrogenase) and glyoxylate shunt (isocitrate lyase and glyoxylate oxidase) responsible for oxalic acid production were estimated. In presence of succinate, phosphate solubilization was completely inhibited, and the enzymes glucose dehydrogenase and glyoxylate oxidase were repressed. Significant activity of isocitrate lyase, the key enzyme for carbon flux through glyoxylate shunt and oxalic acid production during growth on glucose suggested that it could be inducible in nature, and its inhibition by succinate appeared to be similar to catabolite repression.

  3. Interaction of Arabidopsis DET1 with CCA1 and LHY in mediating transcriptional repression in the plant circadian clock

    PubMed Central

    Lau, On Sun; Huang, Xi; Charron, Jean-Benoit; Lee, Jae-Hoon; Li, Gang; Deng, Xing Wang

    2011-01-01

    SUMMARY The COP10-DET1-DDB1 (CDD) complex is an evolutionarily conserved protein complex discovered for its role in the repression of photomorphogenesis in Arabidopsis, and is important in many cellular and developmental processes in both plants and animals. However, its molecular mode of action remains poorly understood. Here, we show that the CDD component, DET1, possesses transcriptional repression activity and physically interacts with two closely related MYB transcription factors, CCA1 and LHY, which are core components of the plant circadian clock. DET1 associates with the promoter of CCA1/LHY target genes, such as TOC1, in a CCA1/LHY-dependent manner and is required for their repression, suggesting a recruitment of DET1 by the central oscillator components to regulate the clock. Our results reveal DET1 as a core transcriptional repression factor important for clock progression. Overall, the CDD complex may function as a transcriptional co-repressor in diverse processes through direct interaction with distinct transcription factors. PMID:21884973

  4. Histone hypoacetylation-activated genes are repressed by acetyl-CoA- and chromatin-mediated mechanism

    PubMed Central

    Mehrotra, Swati; Galdieri, Luciano; Zhang, Tiantian; Zhang, Man; Pemberton, Lucy F.; Vancura, Ales

    2014-01-01

    Transcriptional activation is typically associated with increased acetylation of promoter histones. However, this paradigm does not apply to transcriptional activation of all genes. In this study we have characterized a group of genes that are repressed by histone acetylation. These histone hypoacetylation-activated genes (HHAAG) are normally repressed during exponential growth, when the cellular level of acetyl-CoA is high and global histone acetylation is also high. The HHAAG are induced during diauxic shift, when the levels of acetyl-CoA and global histone acetylation decrease. The histone hypoacetylation-induced activation of HHAAG is independent of Msn2/Msn4. The repression of HSP12, one of the HHAAG, is associated with well-defined nucleosomal structure in the promoter region, while histone hypoacetylation-induced activation correlates with delocalization of positioned nucleosomes or with reduced nucleosome occupancy. Correspondingly, unlike the majority of yeast genes, HHAAG are transcriptionally upregulated when expression of histone genes is reduced. Taken together, these results suggest a model in which histone acetylation is required for proper positioning of promoter nucleosomes and repression of HHAAG. PMID:24907648

  5. Characterization of the functional role of nucleotides within the URE2 IRES element and the requirements for eIF2A-mediated repression.

    PubMed

    Reineke, Lucas C; Merrick, William C

    2009-12-01

    Cap-independent initiation of translation is thought to promote protein synthesis on some mRNAs during times when cap-dependent initiation is down-regulated. However, the mechanism of cap-independent initiation is poorly understood. We have previously reported the secondary structure within the yeast minimal URE2 IRES element. In this study, we sought to investigate the mechanism of internal initiation in yeast by assessing the functional role of nucleotides within the minimal URE2 IRES element, and delineating the cis-sequences that modulate levels of internal initiation using a monocistronic reporter vector. Furthermore, we compared the eIF2A sensitivity of the URE2 IRES element with some of the invasive growth IRES elements using DeltaeIF2A yeast. We found that the stability of the stem-loop structure within the minimal URE2 IRES element is not a critical determinant of optimal IRES activity, and the downstream sequences that modulate URE2 IRES-mediated translation can be defined to discrete regions within the URE2 coding region. Repression of internal initiation on the URE2 minimal IRES element by eIF2A is not dependent on the stability of the secondary structure within the URE2 IRES element. Our data also indicate that eIF2A-mediated repression is not specific to the URE2 IRES element, as both the GIC1 and PAB1 IRES elements are repressed by eIF2A. These data provide valuable insights into the mRNA requirements for internal initiation in yeast, and insights into the mechanism of eIF2A-mediated suppression.

  6. Glucose Repression of STA1 Expression Is Mediated by the Nrg1 and Sfl1 Repressors and the Srb8-11 Complex

    PubMed Central

    Kim, Tae Soo; Lee, Sung Bae; Kang, Hyen Sam

    2004-01-01

    In the yeast Saccharomyces diastaticus, expression of the STA1 gene, which encodes an extracellular glucoamylase, is negatively regulated by glucose. Here we demonstrate that glucose-dependent repression of STA1 expression is imposed by both Sfl1 and Nrg1, which serve as direct transcriptional repressors. We show that Nrg1 acts only on UAS1, and Sfl1 acts only on UAS2, in the STA1 promoter. When bound to its specific site, Sfl1 (but not Nrg1) prevents the binding to UAS2 of two transcriptional activators, Ste12 and Tec1, required for STA1 expression. We also found that Sfl1 contributes to STA1 repression by binding to the promoter and inhibiting the expression of FLO8, a gene that encodes a third transcriptional activator involved in STA1 expression. In addition, we show that the levels of Nrg1 and Sfl1 increase in glucose-grown cells, suggesting that the effects of glucose are mediated, at least in part, through an increase in the abundance of these repressors. NRG1 and SFL1 expression requires the Srb8-11 complex, and correspondingly, the Srb8-11 complex is also necessary for STA1 repression. However, our evidence indicates that the Srb8-11 complex does not associate with either the SFL1 or the NRG1 promoter and thus plays an indirect role in activating NRG1 and SFL1 expression. PMID:15314176

  7. 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

  8. DOF AFFECTING GERMINATION 2 is a positive regulator of light-mediated seed germination and is repressed by DOF AFFECTING GERMINATION 1.

    PubMed

    Santopolo, Silvia; Boccaccini, Alessandra; Lorrai, Riccardo; Ruta, Veronica; Capauto, Davide; Minutello, Emanuele; Serino, Giovanna; Costantino, Paolo; Vittorioso, Paola

    2015-03-04

    The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of the light-mediated seed germination process. DAG1 acts downstream PHYTOCHROME INTERACTING FACTOR3-LIKE 5 (PIL5), the master repressor, and negatively regulates gibberellin biosynthesis by directly repressing the biosynthetic gene AtGA3ox1. The Dof protein DOF AFFECTING GERMINATION (DAG2) shares a high degree of aminoacidic identity with DAG1. While DAG1 inactivation considerably increases the germination capability of seeds, the dag2 mutant has seeds with a germination potential substantially lower than the wild-type, indicating that these factors may play opposite roles in seed germination. We show here that DAG2 expression is positively regulated by environmental factors triggering germination, whereas its expression is repressed by PIL5 and DAG1; by Chromatin Immuno Precipitation (ChIP) analysis we prove that DAG1 directly regulates DAG2. In addition, we show that Red light significantly reduces germination of dag2 mutant seeds. In agreement with the seed germination phenotype of the dag2 mutant previously published, the present data prove that DAG2 is a positive regulator of the light-mediated seed germination process, and particularly reveal that this protein plays its main role downstream of PIL5 and DAG1 in the phytochrome B (phyB)-mediated pathway.

  9. Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame

    PubMed Central

    Park, Hongmarn; McGibbon, Louise C.; Potts, Anastasia H.; Yakhnin, Helen; Romeo, Tony

    2017-01-01

    ABSTRACT CsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of Escherichia coli. Here we identified a novel mechanism in which CsrA represses iraD translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and lacZ fusions demonstrated that CsrA represses iraD expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the iraD leader transcript, all of which are far upstream of the iraD ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the iraD Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the iraD leader region. Two of these ORFs do not affect iraD expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the iraD start codon, a sequence arrangement indicative of translational coupling. In vivo expression and in vitro translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of iraD translation occurs entirely via translational coupling with this ORF, leading to accelerated iraD mRNA decay. PMID:28851853

  10. CLOCKWORK ORANGE Enhances PERIOD Mediated Rhythms in Transcriptional Repression by Antagonizing E-box Binding by CLOCK-CYCLE

    PubMed Central

    Yu, Wangjie; Hardin, Paul E.

    2016-01-01

    The Drosophila circadian oscillator controls daily rhythms in physiology, metabolism and behavior via transcriptional feedback loops. CLOCK-CYCLE (CLK-CYC) heterodimers initiate feedback loop function by binding E-box elements to activate per and tim transcription. PER-TIM heterodimers then accumulate, bind CLK-CYC to inhibit transcription, and are ultimately degraded to enable the next round of transcription. The timing of transcriptional events in this feedback loop coincide with, and are controlled by, rhythms in CLK-CYC binding to E-boxes. PER rhythmically binds CLK-CYC to initiate transcriptional repression, and subsequently promotes the removal of CLK-CYC from E-boxes. However, little is known about the mechanism by which CLK-CYC is removed from DNA. Previous studies demonstrated that the transcription repressor CLOCKWORK ORANGE (CWO) contributes to core feedback loop function by repressing per and tim transcription in cultured S2 cells and in flies. Here we show that CWO rhythmically binds E-boxes upstream of core clock genes in a reciprocal manner to CLK, thereby promoting PER-dependent removal of CLK-CYC from E-boxes, and maintaining repression until PER is degraded and CLK-CYC displaces CWO from E-boxes to initiate transcription. These results suggest a model in which CWO co-represses CLK-CYC transcriptional activity in conjunction with PER by competing for E-box binding once CLK-CYC-PER complexes have formed. Given that CWO orthologs DEC1 and DEC2 also target E-boxes bound by CLOCK-BMAL1, a similar mechanism may operate in the mammalian clock. PMID:27814361

  11. CLOCKWORK ORANGE Enhances PERIOD Mediated Rhythms in Transcriptional Repression by Antagonizing E-box Binding by CLOCK-CYCLE.

    PubMed

    Zhou, Jian; Yu, Wangjie; Hardin, Paul E

    2016-11-01

    The Drosophila circadian oscillator controls daily rhythms in physiology, metabolism and behavior via transcriptional feedback loops. CLOCK-CYCLE (CLK-CYC) heterodimers initiate feedback loop function by binding E-box elements to activate per and tim transcription. PER-TIM heterodimers then accumulate, bind CLK-CYC to inhibit transcription, and are ultimately degraded to enable the next round of transcription. The timing of transcriptional events in this feedback loop coincide with, and are controlled by, rhythms in CLK-CYC binding to E-boxes. PER rhythmically binds CLK-CYC to initiate transcriptional repression, and subsequently promotes the removal of CLK-CYC from E-boxes. However, little is known about the mechanism by which CLK-CYC is removed from DNA. Previous studies demonstrated that the transcription repressor CLOCKWORK ORANGE (CWO) contributes to core feedback loop function by repressing per and tim transcription in cultured S2 cells and in flies. Here we show that CWO rhythmically binds E-boxes upstream of core clock genes in a reciprocal manner to CLK, thereby promoting PER-dependent removal of CLK-CYC from E-boxes, and maintaining repression until PER is degraded and CLK-CYC displaces CWO from E-boxes to initiate transcription. These results suggest a model in which CWO co-represses CLK-CYC transcriptional activity in conjunction with PER by competing for E-box binding once CLK-CYC-PER complexes have formed. Given that CWO orthologs DEC1 and DEC2 also target E-boxes bound by CLOCK-BMAL1, a similar mechanism may operate in the mammalian clock.

  12. Characterization of an Nmr Homolog That Modulates GATA Factor-Mediated Nitrogen Metabolite Repression in Cryptococcus neoformans

    PubMed Central

    Lee, I. Russel; Lim, Jonathan W. C.; Ormerod, Kate L.; Morrow, Carl A.; Fraser, James A.

    2012-01-01

    Nitrogen source utilization plays a critical role in fungal development, secondary metabolite production and pathogenesis. In both the Ascomycota and Basidiomycota, GATA transcription factors globally activate the expression of catabolic enzyme-encoding genes required to degrade complex nitrogenous compounds. However, in the presence of preferred nitrogen sources such as ammonium, GATA factor activity is inhibited in some species through interaction with co-repressor Nmr proteins. This regulatory phenomenon, nitrogen metabolite repression, enables preferential utilization of readily assimilated nitrogen sources. In the basidiomycete pathogen Cryptococcus neoformans, the GATA factor Gat1/Are1 has been co-opted into regulating multiple key virulence traits in addition to nitrogen catabolism. Here, we further characterize Gat1/Are1 function and investigate the regulatory role of the predicted Nmr homolog Tar1. While GAT1/ARE1 expression is induced during nitrogen limitation, TAR1 transcription is unaffected by nitrogen availability. Deletion of TAR1 leads to inappropriate derepression of non-preferred nitrogen catabolic pathways in the simultaneous presence of favoured sources. In addition to exhibiting its evolutionary conserved role of inhibiting GATA factor activity under repressing conditions, Tar1 also positively regulates GAT1/ARE1 transcription under non-repressing conditions. The molecular mechanism by which Tar1 modulates nitrogen metabolite repression, however, remains open to speculation. Interaction between Tar1 and Gat1/Are1 was undetectable in a yeast two-hybrid assay, consistent with Tar1 and Gat1/Are1 each lacking the conserved C-terminus regions present in ascomycete Nmr proteins and GATA factors that are known to interact with each other. Importantly, both Tar1 and Gat1/Are1 are suppressors of C. neoformans virulence, reiterating and highlighting the paradigm of nitrogen regulation of pathogenesis. PMID:22470421

  13. Thyroglobulin repression of thyroid transcription factor 1 (TTF-1) gene expression is mediated by decreased DNA binding of nuclear factor I proteins which control constitutive TTF-1 expression.

    PubMed

    Nakazato, M; Chung, H K; Ulianich, L; Grassadonia, A; Suzuki, K; Kohn, L D

    2000-11-01

    Follicular thyroglobulin (TG) selectively suppresses the expression of thyroid-restricted transcription factors, thereby altering the expression of thyroid-specific proteins. In this study, we investigated the molecular mechanism by which TG suppresses the prototypic thyroid-restricted transcription factor, thyroid transcription factor 1 (TTF-1), in rat FRTL-5 thyrocytes. We show that the region between bp -264 and -153 on the TTF-1 promoter contains two nuclear factor I (NFI) elements whose function is involved in TG-mediated suppression. Thus, NFI binding to these elements is critical for constitutive expression of TTF-1; TG decreases NFI binding to the NFI elements in association with TG repression. NFI is a family of transcription factors that is ubiquitously expressed and contributes to constitutive and cell-specific gene expression. In contrast to the contribution of NFI proteins to constitutive gene expression in other systems, we demonstrate that follicular TG transcriptionally represses all NFI RNAs (NFI-A, -B, -C, and -X) in association with decreased NFI binding and that the RNA levels decrease as early as 4 h after TG treatment. Although TG treatment for 48 h results in a decrease in NFI protein-DNA complexes measured in DNA mobility shift assays, NFI proteins are still detectable by Western analysis. We show, however, that the binding of all NFI proteins is redox regulated. Thus, diamide treatment of nuclear extracts strongly reduces the binding of NFI proteins, and the addition of higher concentrations of dithiothreitol to nuclear extracts from TG-treated cells restores NFI-DNA binding to levels in extracts from untreated cells. We conclude that NFI binding to two NFI elements, at bp -264 to -153, positively regulates TTF-1 expression and controls constitutive TTF-1 levels. TG mediates the repression of TTF-1 gene expression by decreasing NFI RNA and protein levels, as well as by altering the binding activity of NFI, which is redox controlled.

  14. Mechanisms of ceramide-mediated repression of the human telomerase reverse transcriptase promoter via deacetylation of Sp3 by histone deacetylase 1.

    PubMed

    Wooten-Blanks, Leslie G; Song, Pengfei; Senkal, Can E; Ogretmen, Besim

    2007-10-01

    In this study, distinct roles of de novo-generated endogenous ceramides and mechanisms by which deacetylated Sp3 regulates the hTERT promoter activity in response to ceramide signaling were explored. The generation of C18-ceramide via the expression of ceramide synthase 1 (CerS1), and not C16-ceramide by CerS5 or CerS6 expression, resulted in repression of the hTERT promoter via deacetylation of Sp3 by histone deacetylase 1 (HDAC1) in A549 human lung adenocarcinoma cells. Then roles and mechanisms of action of ceramide-mediated deacetylation of Sp3 in inhibiting the hTERT promoter were determined using constitutively deacetylated or acetylated Sp3 mutants at lysine (K) 551. Expression of the deacetylated Sp3 mutant resulted in repression, whereas its acetylated mutant induced basal hTERT promoter activity in Drosophila S2 cells, which do not express any endogenous Sp3, and in A549 cells. Remarkably, chromatin immunoprecipitation data revealed that acetylated Sp3 mutant (K551Q-Sp3) did not bind whereas deacetylated Sp3 (K551R-Sp3) mutant bound strongly to the promoter DNA, resulting in the recruitment of histone deacetylase 1 (HDAC1) and inhibition of the association of RNA polymerase II with the promoter. Mechanistically, increased generation of C18-ceramide by hCerS1 expression, but not by its catalytically inactive mutant, mediated the association and recruitment of the deacetylated Sp3/HDAC1 complex to the hTERT promoter DNA, resulting in the local histone H3 deacetylation and repression of the promoter.

  15. Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame.

    PubMed

    Park, Hongmarn; McGibbon, Louise C; Potts, Anastasia H; Yakhnin, Helen; Romeo, Tony; Babitzke, Paul

    2017-08-29

    CsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of Escherichia coli Here we identified a novel mechanism in which CsrA represses iraD translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and lacZ fusions demonstrated that CsrA represses iraD expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the iraD leader transcript, all of which are far upstream of the iraD ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the iraD Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the iraD leader region. Two of these ORFs do not affect iraD expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the iraD start codon, a sequence arrangement indicative of translational coupling. In vivo expression and in vitro translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of iraD translation occurs entirely via translational coupling with this ORF, leading to accelerated iraD mRNA decay.IMPORTANCE CsrA posttranscriptionally represses gene expression associated with stationary-phase bacterial growth, often in opposition to the transcriptional effects of the stationary-phase sigma factor RpoS. We show that CsrA employs a novel regulatory mechanism to repress translation of iraD, which encodes an

  16. LANA-Mediated Recruitment of Host Polycomb Repressive Complexes onto the KSHV Genome during De Novo Infection

    PubMed Central

    Toth, Zsolt; Papp, Bernadett; Brulois, Kevin; Choi, Youn Jung; Gao, Shou-Jiang; Jung, Jae U.

    2016-01-01

    One of the hallmarks of the latent phase of Kaposi’s sarcoma-associated herpesvirus (KSHV) infection is the global repression of lytic viral gene expression. Following de novo KSHV infection, the establishment of latency involves the chromatinization of the incoming viral genomes and recruitment of the host Polycomb repressive complexes (PRC1 and PRC2) to the promoters of lytic genes, which is accompanied by the inhibition of lytic genes. However, the mechanism of how PRCs are recruited to the KSHV episome is still unknown. Utilizing a genetic screen of latent genes in the context of KSHV genome, we identified the latency-associated nuclear antigen (LANA) to be responsible for the genome-wide recruitment of PRCs onto the lytic promoters following infection. We found that LANA initially bound to the KSHV genome right after infection and subsequently recruited PRCs onto the viral lytic promoters, thereby repressing lytic gene expression. Furthermore, both the DNA and chromatin binding activities of LANA were required for the binding of LANA to the KSHV promoters, which was necessary for the recruitment of PRC2 to the lytic promoters during de novo KSHV infection. Consequently, the LANA-knockout KSHV could not recruit PRCs to its viral genome upon de novo infection, resulting in aberrant lytic gene expression and dysregulation of expression of host genes involved in cell cycle and proliferation pathways. In this report, we demonstrate that KSHV LANA recruits host PRCs onto the lytic promoters to suppress lytic gene expression following de novo infection. PMID:27606464

  17. SUV39H1 mediated SIRT1 trans-repression contributes to cardiac ischemia-reperfusion injury.

    PubMed

    Yang, Guang; Zhang, Xinjian; Weng, Xinyu; Liang, Peng; Dai, Xin; Zeng, Sheng; Xu, Huihui; Huan, Hailin; Fang, Mingming; Li, Yuehua; Xu, Dachun; Xu, Yong

    2017-05-01

    Ischemic reperfusion (I/R) contributes to deleterious cardiac remodeling and heart failure. The deacetylase SIRT1 has been shown to protect the heart from I/R injury. We examined the mechanism whereby I/R injury represses SIRT1 transcription in the myocardium. There was accumulation of trimethylated histone H3K9 on the proximal SIRT1 promoter in the myocardium in mice following I/R injury and in cultured cardiomyocytes exposed to hypoxia-reoxygenation (H/R). In accordance, the H3K9 trimethyltransferase SUV39H1 bound to the SIRT1 promoter and repressed SIRT1 transcription. SUV39H1 expression was up-regulated in the myocardium in mice following I/R insults and in H/R-treated cardiomyocytes paralleling SIRT1 down-regulation. Silencing SUV39H1 expression or suppression of SUV39H1 activity erased H3K9Me3 from the SIRT1 promoter and normalized SIRT1 levels in cardiomyocytes. Meanwhile, SUV39H1 deficiency or inhibition attenuated I/R-induced infarction and improved heart function in mice likely through influencing ROS levels in a SIRT1-dependent manner. Therefore, our data uncover a novel mechanism for SIRT1 trans-repression during cardiac I/R injury and present SUV39H1 as a druggable target for the development of therapeutic strategies against ischemic heart disease.

  18. Pregnane X Receptor (PXR)-Mediated Gene Repression and Cross-Talk of PXR with Other Nuclear Receptors via Coactivator Interactions

    PubMed Central

    Pavek, Petr

    2016-01-01

    Pregnane X receptor is a ligand-activated nuclear receptor (NR) that mainly controls inducible expression of xenobiotics handling genes including biotransformation enzymes and drug transporters. Nowadays it is clear that PXR is also involved in regulation of intermediate metabolism through trans-activation and trans-repression of genes controlling glucose, lipid, cholesterol, bile acid, and bilirubin homeostasis. In these processes PXR cross-talks with other NRs. Accumulating evidence suggests that the cross-talk is often mediated by competing for common coactivators or by disruption of coactivation and activity of other transcription factors by the ligand-activated PXR. In this respect mainly PXR-CAR and PXR-HNF4α interference have been reported and several cytochrome P450 enzymes (such as CYP7A1 and CYP8B1), phase II enzymes (SULT1E1, Gsta2, Ugt1a1), drug and endobiotic transporters (OCT1, Mrp2, Mrp3, Oatp1a, and Oatp4) as well as intermediate metabolism enzymes (PEPCK1 and G6Pase) have been shown as down-regulated genes after PXR activation. In this review, I summarize our current knowledge of PXR-mediated repression and coactivation interference in PXR-controlled gene expression regulation. PMID:27932985

  19. Repression of BIM mediates survival signaling by MYC and AKT in high-risk T-cell acute lymphoblastic leukemia.

    PubMed

    Reynolds, C; Roderick, J E; LaBelle, J L; Bird, G; Mathieu, R; Bodaar, K; Colon, D; Pyati, U; Stevenson, K E; Qi, J; Harris, M; Silverman, L B; Sallan, S E; Bradner, J E; Neuberg, D S; Look, A T; Walensky, L D; Kelliher, M A; Gutierrez, A

    2014-09-01

    Treatment resistance in T-cell acute lymphoblastic leukemia (T-ALL) is associated with phosphatase and tensin homolog (PTEN) deletions and resultant phosphatidylinositol 3'-kinase (PI3K)-AKT pathway activation, as well as MYC overexpression, and these pathways repress mitochondrial apoptosis in established T-lymphoblasts through poorly defined mechanisms. Normal T-cell progenitors are hypersensitive to mitochondrial apoptosis, a phenotype that is dependent on the expression of proapoptotic BIM. In a conditional zebrafish model, MYC downregulation induced BIM expression in T-lymphoblasts, an effect that was blunted by expression of constitutively active AKT. In human T-ALL cell lines and treatment-resistant patient samples, treatment with MYC or PI3K-AKT pathway inhibitors each induced BIM upregulation and apoptosis, indicating that BIM is repressed downstream of MYC and PI3K-AKT in high-risk T-ALL. Restoring BIM function in human T-ALL cells using a stapled peptide mimetic of the BIM BH3 domain had therapeutic activity, indicating that BIM repression is required for T-ALL viability. In the zebrafish model, where MYC downregulation induces T-ALL regression via mitochondrial apoptosis, T-ALL persisted despite MYC downregulation in 10% of bim wild-type zebrafish, 18% of bim heterozygotes and in 33% of bim homozygous mutants (P=0.017). We conclude that downregulation of BIM represents a key survival signal downstream of oncogenic MYC and PI3K-AKT signaling in treatment-resistant T-ALL.

  20. AP-1 mediated relief of repressive activity of the CD30 promoter microsatellite in Hodgkin and Reed-Sternberg cells.

    PubMed

    Watanabe, Mariko; Ogawa, Yuji; Ito, Kinji; Higashihara, Masaaki; Kadin, Marshall E; Abraham, Lawrence J; Watanabe, Toshiki; Horie, Ryouichi

    2003-08-01

    Overexpression of CD30 is the hallmark of Hodgkin and Reed-Sternberg (H-RS) cells and drives constitutive nuclear factor-kappaB activation that is the molecular basis for the pathophysiology of Hodgkin's lymphoma. Transcription of the CD30 gene is controlled by the core promoter that is driven by Sp-1 and the microsatellite sequences (MSs) that represses core promoter activity. To understand the mechanism(s) of CD30 overexpression in H-RS cells, we structurally and functionally characterized the CD30 MSs. Although the CD30 MS of H-RS cell lines was polymorphic, it was not truncated compared with that of control cells. A strong core promoter activity and constitutive Sp-1 binding were revealed in all cell lines examined irrespective of the levels of CD30 expression. In transient reporter gene assays, all MS clones derived from H-RS cell lines repressed the core promoter activity in unrelated cell lines, but not in the H-RS cell lines. An AP-1-binding site was found in the MS at nucleotide position of -377 to -371, the presence of which was found to relieve repression of the core promoter in H-RS cell lines but not in other tumor cell lines. H-RS cell lines showed constitutive and strong AP-1-binding activity, but other cell lines did not. The AP-1 complex contained JunB, whose overexpression activated reporter constructs driven by the CD30 promoter including the MSs, and was dependent on the AP-1 site. JunB expression was detected in H-RS cells in vitro and in vivo, but not in reactive cells or tumor cells of non-Hodgkin's lymphoma of diffuse large B-cell type. Transduction of JunB small interfering RNAs suppressed CD30 promoter activity in L428 cells but not in control cells. Taken together, overexpression and binding of JunB to the AP-1 site appear to relieve the repression of the core promoter by the CD30 MS in H-RS cells, which provide one basis for the constitutive overexpression of CD30 in Hodgkin's lymphoma.

  1. Role of GlnR in acid-mediated repression of genes encoding proteins involved in glutamine and glutamate metabolism in Streptococcus mutans.

    PubMed

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

    2010-04-01

    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 (ATGTNAN(7)TNACAT) 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.

  2. Grb7 Upregulation Is a Molecular Adaptation to HER2 Signaling Inhibition Due to Removal of Akt-Mediated Gene Repression

    PubMed Central

    Nencioni, Alessio; Cea, Michele; Garuti, Anna; Passalacqua, Mario; Raffaghello, Lizzia; Soncini, Debora; Moran, Eva; Zoppoli, Gabriele; Pistoia, Vito; Patrone, Franco; Ballestrero, Alberto

    2010-01-01

    The efficacy of anti-HER2 therapeutics, such as lapatinib and trastuzumab, is limited by primary and acquired resistance. Cellular adaptations that allow breast cancer cell to survive prolonged HER2 inhibition include de-repression of the transcription factor FOXO3A with consequent estrogen receptor activation, and/or increased HER3 signaling. Here, we used low-density arrays, quantitative PCR, and western blotting to determine how HER2 signaling inhibition with lapatinib or PI3K inhibitors affects the expression of genes involved in breast cancer metastatic spread and overall prognosis. Retroviral transgenesis was used to express constitutively active forms of Akt in the HER2+ breast cancer cell line SKBR3, and Grb7 in MCF7 cells. Specific gene silencing was obtained by siRNAs transfection. A murine BT474 xenograft cancer model was used to assess the effect of lapatinib on gene expression in vivo. We found that lapatinib induces upregulation of Grb7, an adaptor protein involved in receptor tyrosine kinase signaling and promoting cell survival and cell migration. Grb7 upregulation induced by lapatinib was found to occur in cancer cells in vitro and in vivo. We demonstrate that Grb7 upregulation is recreated by PI3K inhibitors while being prevented by constitutively active Akt. Thus, Grb7 is repressed by PI3K signaling and lapatinib-mediated Akt inhibition is responsible for Grb7 de-repression. Finally, we show that Grb7 removal by RNA-interference reduces breast cancer cell viability and increases the activity of lapatinib. In conclusion, Grb7 upregulation is a potentially adverse consequence of HER2 signaling inhibition. Preventing Grb7 accumulation and/or its interaction with receptor tyrosine kinases may increase the benefit of HER2-targeting drugs. PMID:20126311

  3. Lateral gene expression in Drosophila early embryos is supported by Grainyhead-mediated activation and tiers of dorsally-localized repression.

    PubMed

    Garcia, Mayra; Stathopoulos, Angelike

    2011-01-01

    The general consensus in the field is that limiting amounts of the transcription factor Dorsal establish dorsal boundaries of genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, while repressors establish ventral boundaries. Yet recent studies have provided evidence that repressors act to specify the dorsal boundary of intermediate neuroblasts defective (ind), a gene expressed in a stripe along the DV axis in lateral regions of the embryo. Here we show that a short 12 base pair sequence ("the A-box") present twice within the ind CRM is both necessary and sufficient to support transcriptional repression in dorsal regions of embryos. To identify binding factors, we conducted affinity chromatography using the A-box element and found a number of DNA-binding proteins and chromatin-associated factors using mass spectroscopy. Only Grainyhead (Grh), a CP2 transcription factor with a unique DNA-binding domain, was found to bind the A-box sequence. Our results suggest that Grh acts as an activator to support expression of ind, which was surprising as we identified this factor using an element that mediates dorsally-localized repression. Grh and Dorsal both contribute to ind transcriptional activation. However, another recent study found that the repressor Capicua (Cic) also binds to the A-box sequence. While Cic was not identified through our A-box affinity chromatography, utilization of the same site, the A-box, by both factors Grh (activator) and Cic (repressor) may also support a "switch-like" response that helps to sharpen the ind dorsal boundary. Furthermore, our results also demonstrate that TGF-β signaling acts to refine ind CRM expression in an A-box independent manner in dorsal-most regions, suggesting that tiers of repression act in dorsal regions of the embryo.

  4. c-Myc represses FOXO3a-mediated transcription of the gene encoding the p27(Kip1) cyclin dependent kinase inhibitor.

    PubMed

    Chandramohan, Vidyalakshmi; Mineva, Nora D; Burke, Brian; Jeay, Sébastien; Wu, Min; Shen, Jian; Yang, William; Hann, Stephen R; Sonenshein, Gail E

    2008-08-15

    The p27(Kip1) (p27) cyclin-dependent kinase inhibitor and c-Myc oncoprotein play essential roles in control of cell cycle progression and apoptosis. Induction of p27 (CDKN1B) gene transcription by Forkhead box O proteins such as FOXO3a leads to growth arrest and apoptosis. Previously, we observed that B cell receptor (surface IgM) engagement of WEHI 231 immature B lymphoma cells with an anti-IgM antibody results in activation of FOXO3a, growth arrest and apoptosis. As ectopic c-Myc expression in these cells prevented anti-IgM induction of p27 and cell death, we hypothesized that c-Myc represses FOXO3a-mediated transcription. Here we show that c-Myc inhibits FOXO3a-mediated activation of the p27 promoter in multiple cell lines. The mechanism of this repression was explored using a combination of co-immunoprecipitation, oligonucleotide precipitation, and chromatin immunoprecipitation experiments. The studies demonstrate a functional association of FOXO3a and c-Myc on a proximal Forkhead binding element in the p27 promoter. This association involves the Myc box II domain of c-Myc and the N-terminal DNA-binding portion of FOXO3a. Analysis of publicly available microarray datasets showed an inverse pattern of c-MYC and p27 RNA expression in primary acute myeloid leukemia, prostate cancer and tongue squamous cell carcinoma samples. The inhibition of FOXO3a-mediated activation of the p27 gene by the high aberrant expression of c-Myc in many tumor cells likely contributes to their uncontrolled proliferation and invasive phenotype.

  5. Ezh2-mediated repression of a transcriptional pathway upstream of Mmp9 maintains integrity of the developing vasculature

    PubMed Central

    Delgado-Olguín, Paul; Dang, Lan T.; He, Daniel; Thomas, Sean; Chi, Lijun; Sukonnik, Tatyana; Khyzha, Nadiya; Dobenecker, Marc-Werner; Fish, Jason E.; Bruneau, Benoit G.

    2014-01-01

    Maintenance of vascular integrity is required for embryogenesis and organ homeostasis. However, the gene expression programs that stabilize blood vessels are poorly understood. Here, we show that the histone methyltransferase Ezh2 maintains integrity of the developing vasculature by repressing a transcriptional program that activates expression of Mmp9. Inactivation of Ezh2 in developing mouse endothelium caused embryonic lethality with compromised vascular integrity and increased extracellular matrix degradation. Genome-wide approaches showed that Ezh2 targets Mmp9 and its activators Fosl1 and Klf5. In addition, we uncovered Creb3l1 as an Ezh2 target that directly activates Mmp9 gene expression in the endothelium. Furthermore, genetic inactivation of Mmp9 rescued vascular integrity defects in Ezh2-deficient embryos. Thus, epigenetic repression of Creb3l1, Fosl1, Klf5 and Mmp9 by Ezh2 in endothelial cells maintains the integrity of the developing vasculature, potentially linking this transcriptional network to diseases with compromised vascular integrity. PMID:25359725

  6. Small Ubiquitin-like Modifier (SUMO)-mediated Repression of the Xenopus Oocyte 5 S rRNA Genes*

    PubMed Central

    Malik, Mariam Q.; Bertke, Michelle M.; Huber, Paul W.

    2014-01-01

    The 5 S rRNA gene-specific transcription factor IIIA (TFIIIA) interacts with the small ubiquitin-like modifier (SUMO) E3 ligase PIAS2b and with one of its targets, the transcriptional corepressor, XCtBP. PIAS2b is restricted to the cytoplasm of Xenopus oocytes but relocates to the nucleus immediately after fertilization. Following the midblastula transition, PIAS2b and XCtBP are present on oocyte-type, but not somatic-type, 5 S rRNA genes up through the neurula stage, as is a limiting amount of TFIIIA. Histone H3 methylation, coincident with the binding of XCtBP, also occurs exclusively on the oocyte-type genes. Immunohistochemical staining of embryos confirms the occupancy of a subset of the oocyte-type genes by TFIIIA that become positioned at the nuclear periphery shortly after the midblastula transition. Inhibition of SUMOylation activity relieves repression of oocyte-type 5 S rRNA genes and is correlated with a decrease in methylation of H3K9 and H3K27 and disruption of subnuclear localization. These results reveal a novel function for TFIIIA as a negative regulator that recruits histone modification activity through the CtBP repressor complex exclusively to the oocyte-type 5 S rRNA genes, leading to their terminal repression. PMID:25368327

  7. Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1

    DOE PAGES

    Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany; ...

    2017-01-31

    The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

  8. Nuclear localization of MBNL1: splicing-mediated autoregulation and repression of repeat-derived aberrant proteins.

    PubMed

    Kino, Yoshihiro; Washizu, Chika; Kurosawa, Masaru; Oma, Yoko; Hattori, Nobutaka; Ishiura, Shoichi; Nukina, Nobuyuki

    2015-02-01

    In some neurological diseases caused by repeat expansions such as myotonic dystrophy, the RNA-binding protein muscleblind-like 1 (MBNL1) accumulates in intranuclear inclusions containing mutant repeat RNA. The interaction between MBNL1 and mutant RNA in the nucleus is a key event leading to loss of MBNL function, yet the details of this effect have been elusive. Here, we investigated the mechanism and significance of MBNL1 nuclear localization. We found that MBNL1 contains two classes of nuclear localization signal (NLS), a classical bipartite NLS and a novel conformational NLS. Alternative splicing of exon 7 acts as a switch between these NLS types and couples MBNL1 activity and intracellular localization. Depending on its nuclear localization, MBNL1 promoted nuclear accumulation of mutant RNA containing a CUG or CAG repeat, some of which produced proteins containing homopolymeric tracts such as polyglutamine. Furthermore, MBNL1 repressed the expression of these homopolymeric proteins including those presumably produced through repeat-associated non-ATG (RAN) translation. These results suggest that nuclear retention of expanded RNA reflects a novel role of MBNL proteins in repressing aberrant protein expression and may provide pathological and therapeutic implications for a wide range of repeat expansion diseases associated with nuclear RNA retention and/or RAN translation.

  9. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria.

    PubMed

    Willmann, Anita; Weiss, Michael; Nehls, Uwe

    2007-02-01

    A main function of ectomycorrhizas, a symbiosis between certain soil fungi and fine roots of woody plants, is the exchange of plant-derived carbohydrates for fungus-derived nutrients. As it is required in large amounts, nitrogen is of special interest. A gene (AmAMT2) coding for a putative fungal ammonium importer was identified in an EST project of functional Amanita muscaria/poplar ectomycorrhizas. Heterologous expression of the entire AmAMT2 coding region in yeast revealed the corresponding protein to be a high-affinity ammonium importer. In axenically grown Amanita hyphae AmAMT2 expression was strongly repressed by nitrogen, independent of whether the offered nitrogen source was transported by AmAMT2 or not. In functional ectomycorrhizas the AmAMT2 transcript level was further decreased in both hyphal networks (sheath and Hartig net), while extraradical hyphae revealed strong gene expression. Together our data suggest that (1) AmAMT2 expression is regulated by the endogenous nitrogen content of hyphae and (2) fungal hyphae in ectomycorrhizas are well supported with nitrogen even when the extraradical mycelium is nitrogen limited. As a consequence of AmAMT2 repression in mycorrhizas, ammonium can be suggested as a potential nitrogen source delivered by fungal hyphae in symbiosis.

  10. WT1-mediated repression of the proapoptotic transcription factor ZNF224 is triggered by the BCR-ABL oncogene

    PubMed Central

    Montano, Giorgia; Vidovic, Karina; Palladino, Chiara; Cesaro, Elena; Sodaro, Gaetano; Quintarelli, Concetta; De Angelis, Biagio; Errichiello, Santa; Pane, Fabrizio; Izzo, Paola; Grosso, Michela; Gullberg, Urban; Costanzo, Paola

    2015-01-01

    The Kruppel-like protein ZNF224 is a co-factor of the Wilms’ tumor 1 protein, WT1. We have previously shown that ZNF224 exerts a specific proapoptotic role in chronic myelogenous leukemia (CML) K562 cells and contributes to cytosine arabinoside-induced apoptosis, by modulating WT1-dependent transcription of apoptotic genes. Here we demonstrate that ZNF224 gene expression is down-regulated both in BCR-ABL positive cell lines and in primary CML samples and is restored after imatinib and second generation tyrosine kinase inhibitors treatment. We also show that WT1, whose expression is positively regulated by BCR-ABL, represses transcription of the ZNF224 gene. Finally, we report that ZNF224 is significantly down-regulated in patients with BCR-ABL positive chronic phase-CML showing poor response or resistance to imatinib treatment as compared to high-responder patients. Taken as a whole, our data disclose a novel pathway activated by BCR-ABL that leads to inhibition of apoptosis through the ZNF224 repression. ZNF224 could thus represent a novel promising therapeutic target in CML. PMID:26320177

  11. 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

  12. Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1

    PubMed Central

    Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany; Sandate, Colby R.; Hura, Greg L.; Schneidman-Duhovny, Dina; Takahashi, Joseph S.; Partch, Carrie L.

    2017-01-01

    The basic helix–loop–helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ∼24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here we show that CRY1 binds directly to the PAS domain core of CLOCK:BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solution X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1. PMID:28143926

  13. 15-Deoxy-Δ12,14-prostaglandin J2-Glycerol Ester, a Putative Metabolite of 2-Arachidonyl Glycerol, Activates Peroxisome Proliferator Activated Receptor γ

    PubMed Central

    Raman, Priyadarshini; Kaplan, Barbara L. F.; Thompson, Jerry T.; Vanden Heuvel, John P.

    2011-01-01

    2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative capable of suppressing interleukin (IL)-2 production by activated T cells. 2-AG-mediated IL-2 suppression is dependent on cyclooxygenase-2 (COX-2) metabolism and peroxisome proliferator activated receptor γ (PPARγ) activation. The objective of the present studies was to examine whether 15-deoxy-Δ12,14-PGJ2-glycerol ester (15d-PGJ2-G), a putative metabolite of 2-AG, can mimic the actions of 2-AG on IL-2 regulation through PPARγ activation. 15d-PGJ2-G bound PPARγ-ligand binding domain in a PPARγ competitive binding assay. 15d-PGJ2-G treatment activated PPARγ in a reporter assay, and PPARγ activation was attenuated when a PPARγ antagonist, 2-chloro-5-nitro-N-4-pyridinylbenzamide (T0070907), was present. 15d-PGJ2-G treatment suppressed IL-2 production by activated Jurkat cells, which was partially attenuated when pretreated with T0070907. Moreover, IL-2 suppression was pronounced when 15d-PGJ2-G was present 30 min before or after T-cell activation. Concordant with IL-2 suppression, 15d-PGJ2-G treatment decreased nuclear factor of activated T cells (NFAT) transcriptional activity in transiently transfected Jurkat cells. It is noteworthy that T0070907 alone markedly increased NFAT reporter activity, suggesting the existence of endogenous PPARγ activation and modulation of NFAT. Because COX-2 metabolism of 2-AG is important for IL-2 suppression, the effect of 2-AG on COX-2 and PPARγ mRNA expression was investigated. 2-AG treatment decreased the up-regulation of COX-2 mRNA after T-cell activation, which suggests negative feedback limiting COX-2-mediated metabolism of 2-AG. PPARγ mRNA expression was increased upon activation, and 2-AG treatment produced a modest decrease in PPARγ mRNA expression. Collectively, our findings suggest that 15d-PGJ2-G activates PPARγ to decrease NFAT transcriptional activity and IL-2 expression in activated T cells. PMID:21511917

  14. 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.

  15. The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger.

    PubMed Central

    Kudla, B; Caddick, M X; Langdon, T; Martinez-Rossi, N M; Bennett, C F; Sibley, S; Davies, R W; Arst, H N

    1990-01-01

    The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans has been sequenced and its transcript mapped and orientated. A single ORF can encode a protein of 719 amino acids. A 52 amino acid region including a putative 'zinc finger' strongly resembles putative DNA binding regions of the major regulatory protein of erythroid cells. The derived protein sequence also contains a highly acidic region possibly involved in gene activation and 22 copies of the motif S(T)PXX, abundant in DNA binding proteins. Analysis of chromosomal rearrangements and transformation with deletion clones identified 342 N-terminal and 124 C-terminal residues as inessential and localized a C-terminal region required for nitrogen metabolite repressibility. A -1 frameshift eliminating the inessential 122 C-terminal amino acids is a surprising loss-of-function mutation. Extraordinary basicity of the replacement C terminus might explain its phenotype. Mutant sequencing also identified a polypeptide chain termination and several missense mutations, but most interesting are sequence changes associated with specificity mutations. A mutation elevating expression of some structural genes under areA control whilst reducing or not affecting expression of others is a leucine to valine change in the zinc finger loop. It reverts to a partly reciprocal phenotype by replacing the mutant valine by methionine. Images Fig.2 Fig.4 Fig.5 Fig. 8. Fig. 9. PMID:1970293

  16. REDD1 enhances protein phosphatase 2A-mediated dephosphorylation of Akt to repress mTORC1 signaling

    PubMed Central

    Dennis, Michael D.; Coleman, Catherine S.; Berg, Arthur; Jefferson, Leonard S.; Kimball, Scot R.

    2014-01-01

    The protein kinase mTOR (mechanistic target of rapamycin) in complex 1 (mTORC1) promotes cell growth and proliferation in response to anabolic stimuli, including growth factors and nutrients. Growth factors activate mTORC1 by stimulating the kinase Akt, which phosphorylates and inhibits the tuberous sclerosis complex (TSC; which is comprised of TSC1, TSC2, and TBC1D7), thereby stimulating the mTORC1 activator Rheb. Here, we identified the mechanism through which REDD1 (regulated in DNA damage and development 1) represses the mTORC1 signaling pathway. We found that REDD1 promoted the protein phosphatase 2A (PP2A)-dependent dephosphorylation of Akt at Thr308 but not at Ser473. Consistent with previous studies showing that phosphorylation of Akt on Thr308, but not Ser473, is necessary for phosphorylation of TSC2, we observed a REDD1-dependent reduction in the phosphorylation of TSC2 and subsequently in the activity of Rheb. REDD1 and PP2A coimmunoprecipitated with Akt from wild-type but not REDD1-knockout mouse embryonic fibroblasts, suggesting that REDD1 may act as a targeting protein for the catalytic subunit of PP2A. Furthermore, binding to both Akt and PP2A was essential for REDD1 to repress signaling to mTORC1. Overall, the results demonstrate that REDD1 acts not just as a repressor of mTORC1, but also as a constant modulator of the phosphorylation of Akt in response to growth factors and nutrients. PMID:25056877

  17. Epoxide-mediated CifR repression of cif gene expression utilizes two binding sites in Pseudomonas aeruginosa.

    PubMed

    Ballok, Alicia E; Bahl, Christopher D; Dolben, Emily L; Lindsay, Allia K; St Laurent, Jessica D; Hogan, Deborah A; Madden, Dean R; O'Toole, George A

    2012-10-01

    Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro, and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees.

  18. Epoxide-Mediated CifR Repression of cif Gene Expression Utilizes Two Binding Sites in Pseudomonas aeruginosa

    PubMed Central

    Ballok, Alicia E.; Bahl, Christopher D.; Dolben, Emily L.; Lindsay, Allia K.; St. Laurent, Jessica D.; Hogan, Deborah A.; Madden, Dean R.

    2012-01-01

    Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the −10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro, and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees. PMID:22843844

  19. 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

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

    PubMed Central

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

    2001-01-01

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

  1. Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-{Delta}{sup 12,14}-prostaglandin J2

    SciTech Connect

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

    2010-04-23

    Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 {mu}M concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTP{gamma}S or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.

  2. Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-Delta(12,14)-prostaglandin J2.

    PubMed

    Mishra, Nandita; Kar, Rekha; Singha, Prajjal K; Venkatachalam, Manjeri A; McEwen, Donald G; Saikumar, Pothana

    2010-04-23

    Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 microM concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTPgammaS or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.

  3. Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 Deoxy- Δ12,14 - Prostaglandin J2

    PubMed Central

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

    2010-01-01

    Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1µM concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTPγS or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration. PMID:20307494

  4. Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways.

    PubMed

    Jullien, Jerome; Vodnala, Munender; Pasque, Vincent; Oikawa, Mami; Miyamoto, Kei; Allen, George; David, Sarah Anne; Brochard, Vincent; Wang, Stan; Bradshaw, Charles; Koseki, Haruhiko; Sartorelli, Vittorio; Beaujean, Nathalie; Gurdon, John

    2017-03-02

    Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.

  5. Su(H)-Mediated Repression Positions Gene Boundaries along the Dorsal-Ventral Axis of Drosophila Embryos

    PubMed Central

    Ozdemir, Anil; Ma, Lijia; White, Kevin P.; Stathopoulos, Angelike

    2014-01-01

    SUMMARY In Drosophila embryos, a nuclear gradient of the Dorsal transcription factor directs differential gene expression along the dorsoventral (DV) axis, translating it into distinct domains that specify future mesodermal, neural, and ectodermal territories. However, the mechanisms used to differentially-position gene expression boundaries along this axis are not fully understood. Here using a combination of approaches including mutant phenotype analyses and chromatin-immunoprecipitation, we show that the transcription factor Suppressor of Hairless [Su(H)] helps define dorsal boundaries for many genes expressed along the DV axis. Synthetic reporter constructs also provide molecular evidence that Su(H) binding sites support repression and act to counterbalance activation through Dl and the ubiquitous activator Zelda. Our study highlights a role for broadly-expressed repressors, like Su(H), and organization of transcription factor binding sites within cis-regulatory modules as important elements controlling spatial domains of gene expression, to facilitate flexible positioning of boundaries across the entire DV axis. PMID:25313963

  6. Su(H)-mediated repression positions gene boundaries along the dorsal-ventral axis of Drosophila embryos.

    PubMed

    Ozdemir, Anil; Ma, Lijia; White, Kevin P; Stathopoulos, Angelike

    2014-10-13

    In Drosophila embryos, a nuclear gradient of the Dorsal (Dl) transcription factor directs differential gene expression along the dorsoventral (DV) axis, translating it into distinct domains that specify future mesodermal, neural, and ectodermal territories. However, the mechanisms used to differentially position gene expression boundaries along this axis are not fully understood. Here, using a combination of approaches, including mutant phenotype analyses and chromatin immunoprecipitation, we show that the transcription factor Suppressor of Hairless, Su(H), helps define dorsal boundaries for many genes expressed along the DV axis. Synthetic reporter constructs also provide molecular evidence that Su(H) binding sites support repression and act to counterbalance activation through Dl and the ubiquitous activator Zelda. Our study highlights a role for broadly expressed repressors, like Su(H), and organization of transcription factor binding sites within cis-regulatory modules as important elements controlling spatial domains of gene expression to facilitate flexible positioning of boundaries across the entire DV axis. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. 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

  8. KLF4 regulates adult lung tumor-initiating cells and represses K-Ras-mediated lung cancer.

    PubMed

    Yu, T; Chen, X; Zhang, W; Liu, J; Avdiushko, R; Napier, D L; Liu, A X; Neltner, J M; Wang, C; Cohen, D; Liu, C

    2016-02-01

    Lung cancer is the leading cause of cancer-related mortality in both men and women worldwide. To identify novel factors that contribute to lung cancer pathogenesis, we analyzed a lung cancer database from The Cancer Genome Atlas and found that Krüppel-like Factor 4 (KLF4) expression is significantly lower in patients' lung cancer tissue than in normal lung tissue. In addition, we identified seven missense mutations in the KLF4 gene. KLF4 is a transcription factor that regulates cell proliferation and differentiation as well as the self-renewal of stem cells. To understand the role of KLF4 in the lung, we generated a tamoxifen-induced Klf4 knockout mouse model. We found that KLF4 inhibits lung cancer cell growth and that depletion of Klf4 altered the differentiation pattern in the developing lung. To understand how KLF4 functions during lung tumorigenesis, we generated the K-ras(LSL-G12D/+);Klf4(fl/fl) mouse model, and we used adenovirus-expressed Cre to induce K-ras activation and Klf4 depletion in the lung. Although Klf4 deletion alone or K-ras mutation alone can trigger lung tumor formation, Klf4 deletion combined with K-ras mutation significantly enhanced lung tumor formation. We also found that Klf4 deletion in conjunction with K-ras activation caused lung inflammation. To understand the mechanism whereby KLF4 is regulated during lung tumorigenesis, we analyzed KLF4 promoter methylation and the profiles of epigenetic factors. We found that Class I histone deacetylases (HDACs) are overexpressed in lung cancer and that HDAC inhibitors induced expression of KLF4 and inhibited proliferation of lung cancer cells, suggesting that KLF4 is probably repressed by histone acetylation and that HDACs are valuable drug targets for lung cancer treatment.

  9. Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2

    PubMed Central

    Liwak, Urszula; Thakor, Nehal; Jordan, Lindsay E.; Roy, Rajat; Lewis, Stephen M.; Pardo, Olivier E.; Seckl, Michael

    2012-01-01

    Apoptosis can be regulated by extracellular signals that are communicated by peptides such as fibroblast growth factor 2 (FGF-2) that have important roles in tumor cell proliferation. The prosurvival effects of FGF-2 are transduced by the activation of the ribosomal protein S6 kinase 2 (S6K2), which increases the expression of the antiapoptotic proteins X chromosome-linked Inhibitor of Apoptosis (XIAP) and Bcl-xL. We now show that the FGF-2–S6K2 prosurvival signaling is mediated by the tumor suppressor programmed cell death 4 (PDCD4). We demonstrate that PDCD4 specifically binds to the internal ribosome entry site (IRES) elements of both the XIAP and Bcl-xL messenger RNAs and represses their translation by inhibiting the formation of the 48S translation initiation complex. Phosphorylation of PDCD4 by activated S6K2 leads to the degradation of PDCD4 and thus the subsequent derepression of XIAP and Bcl-xL translation. Our results identify PDCD4 as a specific repressor of the IRES-dependent translation of cellular mRNAs (such as XIAP and Bcl-xL) that mediate FGF-2–S6K2 prosurvival signaling and provide further insight into the role of PDCD4 in tumor suppression. PMID:22431522

  10. KMT1E mediated H3K9 methylation is required for the maintenance of embryonic stem cells by repressing trophectoderm differentiation.

    PubMed

    Lohmann, Felix; Loureiro, Joseph; Su, Hui; Fang, Qing; Lei, Hong; Lewis, Tanya; Yang, Yi; Labow, Mark; Li, En; Chen, Taiping; Kadam, Shilpa

    2010-02-01

    Dynamic regulation of histone methylation by methyltransferases and demethylases plays a central role in regulating the fate of embryonic stem (ES) cells. The histone H3K9 methyltransferase KMT1E, formerly known as ESET or Setdb1, is essential to embryonic development as the ablation of the Setdb1 gene results in peri-implantation lethality and prevents the propagation of ES cells. However, Setdb1-null blastocysts do not display global changes in H3K9 methylation or DNA methylation, arguing against a genome-wide defect. Here we show that conditional deletion of the Setdb1 gene in ES cells results in the upregulation of lineage differentiation markers, especially trophectoderm-specific factors, similar to effects observed upon loss of Oct3/4 expression in ES cells. We demonstrate that KMT1E deficiency in ES cells leads to a decrease in histone H3K9 methylation at and derepression of trophoblast-associated genes such as Cdx2. Furthermore, we find genes that are derepressed upon Setdb1 deletion to overlap with known targets of polycomb mediated repression, suggesting that KMT1E mediated H3K9 methylation acts in concert with polycomb controlled H3K27 methylation. Our studies thus demonstrate an essential role for KMT1E in the control of developmentally regulated gene expression programs in ES cells.

  11. 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.

  12. 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.

  13. Copper metabolism domain-containing 1 represses the mediators involved in the terminal effector pathways of human labour and delivery.

    PubMed

    Lappas, Martha

    2016-04-01

    Does Copper Metabolism MURR1 Domain 1 (COMMD1) play a role in regulating the mediators involved in the terminal processes of human labour and delivery? COMMD1 plays a critical role in the termination of nuclear factor-κB (NF-κB) activity and the control of pro-inflammatory and pro-labour mediators. Inflammation and infection are the biggest aetiological factors associated with preterm birth. NF-κB drives the transcription of pro-inflammatory mediators involved in the terminal effector pathways of human labour and delivery. In non-gestational tissues, COMMD1 is a negative regulator of NF-κB-induced inflammation. The mRNA and/or protein level of COMMD1 was assessed in myometrium (n = 8 per group) and fetal membranes (n = 8 per group) obtained from term non-labouring and labouring women at term, and fetal membranes (n = 8 per group) at preterm with and without histological chorioamnionitis. Primary human myometrial cells were used to determine the effect of pro-inflammatory mediators on COMMD1 level, and the effect of COMMD1 small interfering RNA (siRNA) on pro-labour mediators. Statistical significance was ascribed to a P < 0.05. COMMD1 expression was significantly decreased with spontaneous term labour in myometrium; in fetal membranes with histologically confirmed chorioamnionitis and in myometrial cells treated with pro-inflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, the bacterial product fibroblast-stimulating lipopeptide and the viral double stranded RNA analogue polyinosinic polycytidilic acid. Loss-of-function studies revealed an increase in inflammation- and infection-induced TNF-α, IL-1α, IL-1β, IL-6, IL-8 and/or monocyte chemoattractant protein-1 mRNA abundance and/or release; and cyclo-oxygenase-2 mRNA level, release of prostaglandin (PG) F2α and mRNA level of the PGF2α receptor FP. In addition, siRNA knockdown of COMMD1 was associated with significantly increased NF-κB activation as evidenced by increased IL-1

  14. Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation

    PubMed Central

    Várallyay, Éva; Válóczi, Anna; Ágyi, Ákos; Burgyán, József; Havelda, Zoltán

    2010-01-01

    Virus infections induce the expression of ARGONAUTE1 (AGO1) mRNA and in parallel enhance the accumulation of miR168 (regulator of AGO1 mRNA). Here, we show that in virus-infected plants the enhanced expression of AGO1 mRNA is not accompanied by increased AGO1 protein accumulation. We also show that the induction of AGO1 mRNA level is a part of the host defence reaction, whereas the induction of miR168, which overlaps spatially with virus-occupied sectors, is mediated mainly by the Tombusvirus p19 RNA-silencing suppressor. The absence of p19 results in the elimination of miR168 induction and accompanied with the enhanced accumulation of AGO1 protein. In transient expression study, p19 mediates the induction of miR168 and the down-regulation of endogenous AGO1 level. P19 is not able to efficiently bind miR168 in virus-infected plants, indicating that this activity is uncoupled from the small RNA-binding capacity of p19. Our results imply that plant viruses can inhibit the translational capacity of AGO1 mRNA by modulating the endogenous miR168 level to alleviate the anti-viral function of AGO1 protein. PMID:20823831

  15. Restoration of MYC-repressed targets mediates the negative effects of GM-CSF on RUNX1-ETO leukemogenicity.

    PubMed

    Weng, S; Matsuura, S; Mowery, C T; Stoner, S A; Lam, K; Ran, D; Davis, A G; Lo, M-C; Zhang, D-E

    2017-01-01

    Granulocyte macrophage-colony-stimulating factor (GM-CSF) signaling regulates hematopoiesis and immune responses. CSF2RA, the gene encoding the α-subunit for GM-CSF, is significantly downregulated in t(8;21) (RUNX1-ETO or RE) leukemia patients, suggesting that it may serve as a tumor suppressor. We previously reported that GM-CSF signaling is inhibitory to RE leukemogenesis. Here we conducted gene expression profiling of primary RE hematopoietic stem/progenitor cells (HSPCs) treated with GM-CSF to elucidate the mechanisms mediating the negative effects of GM on RE leukemogenicity. We observed that GM treatment of RE HSPCs resulted in a unique gene expression profile that resembles primary human cells undergoing myelopoiesis, which was not observed in control HSPCs. Additionally, we discovered that GM-CSF signaling attenuates MYC-associated gene signatures in RE HSPCs. In agreement with this, a functional screen of a subset of GM-CSF-responsive genes demonstrated that a MYC inhibitor, MXI1 (Max interactor 1), reduced the leukemic potential of RE HSPCs and t(8;21) acute myeloid leukemia (AML) cells. Furthermore, MYC knockdown and treatment with the BET (bromodomain and extra terminal domain) inhibitor JQ1 reduced the leukemic potential of t(8;21) cell lines. Altogether, we discovered a novel molecular mechanism mediating the GM-CSF-induced reduction in leukemic potential of RE cells, and our findings support MYC inhibition as an effective strategy for reducing the leukemogenicity of t(8;21) AML.

  16. Genetic Interactions Between P Elements Involved in piRNA-Mediated Repression of Hybrid Dysgenesis in Drosophila melanogaster

    PubMed Central

    Simmons, Michael J.; Meeks, Marshall W.; Jessen, Erik; Becker, Jordan R.; Buschette, Jared T.; Thorp, Michael W.

    2014-01-01

    Previous studies have shown that telomeric P elements inserted at the left end of the X chromosome are anchors of the P cytotype, the maternally inherited state that regulates P-element activity in the germ line of Drosophila melanogaster. This regulation is mediated by small RNAs that associate with the Piwi family of proteins (piRNAs). We extend the analysis of cytotype regulation by studying new combinations of telomeric and nontelomeric P elements (TPs and non-TPs). TPs interact with each other to enhance cytotype regulation. This synergism involves a strictly maternal effect, called presetting, which is apparently mediated by piRNAs transmitted through the egg. Presetting by a maternal TP can elicit regulation by an inactive paternally inherited TP, possibly by stimulating its production of primary piRNAs. When one TP has come from a stock heterozygous for a mutation in the aubergine, piwi, or Suppressor of variegation 205 genes, the synergism between two TPs is impaired. TPs also interact with non-TPs to enhance cytotype regulation, even though the non-TPs lack regulatory ability on their own. Non-TPs are not susceptible to presetting by a TP, nor is a TP susceptible to presetting by a non-TP. The synergism between TPs and non-TPs is stronger when the TP was inherited maternally. This synergism may be due to the accumulation of secondary piRNAs created by ping-pong cycling between primary piRNAs from the TPs and mRNAs from the non-TPs. Maternal transmission of P-element piRNAs plays an important role in the maintenance of strong cytotype regulation over generations. PMID:24902606

  17. 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.

  18. 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.

  19. ERα-mediated repression of pro-inflammatory cytokine expression by glucocorticoids reveals a crucial role for TNFα and IL1α in lumen formation and maintenance

    PubMed Central

    Eritja, Nuria; Mirantes, Cristina; Llobet, David; Masip, Gemma; Matias-Guiu, Xavier; Dolcet, Xavi

    2012-01-01

    Most glandular tissues comprise polarized epithelial cells organized around a single central lumen. Although there is active research investigating the molecular networks involved in the regulation of lumenogenesis, little is known about the extracellular factors that influence lumen formation and maintenance. Using a three-dimensional culture system of epithelial endometrial cells, we have revealed a new role for pro-inflammatory cytokines such as TNFα and IL1α in the formation and, more importantly, maintenance of a single central lumen. We also studied the mechanism by which glucocorticoids repress TNFα and IL1α expression. Interestingly, regulation of pro-inflammatory cytokine expression and subsequent lumen formation is mediated by estrogen receptor α (ERα) but not by the glucocorticoid receptor. Finally, we investigated the signaling pathways involved in the regulation of lumen formation by pro-inflammatory cytokines. Our results demonstrate that activation of the ERK/MAPK signaling pathway, but not the PI3K/Akt signaling pathway, is important for the formation and maintenance of a single central lumen. In summary, our results suggest a novel role for ERα-regulated pro-inflammatory cytokine expression in lumen formation and maintenance. PMID:22328525

  20. A new regulatory element mediates ethanol repression of KlADH3, a Kluyveromyces lactis gene coding for a mitochondrial alcohol dehydrogenase.

    PubMed

    Saliola, Michele; Getuli, Claudia; Mazzoni, Cristina; Fantozzi, Ivana; Falcone, Claudio

    2007-08-01

    KlADH3 is a Kluyveromyces lactis alcohol dehydrogenase gene induced in the presence of all respiratory carbon sources except ethanol, which specifically represses this gene. Deletion analysis of the KlADH3 promoter revealed the presence of both positive and negative elements. However, by site-directed mutagenesis and gel retardation experiments, we identified a 15-bp element responsible for the transcriptional repression of this gene by ethanol. In particular, this element showed putative sites required for the sequential binding of ethanol-induced factors responsible for the repressed conditions, and the binding of additional factors relieved repression. In addition, we showed that the ethanol element was required for in vivo repression of KlAdh3 activity.

  1. Repression of c-Kit by p53 is mediated by miR-34 and is associated with reduced chemoresistance, migration and stemness.

    PubMed

    Siemens, Helge; Jackstadt, Rene; Kaller, Markus; Hermeking, Heiko

    2013-09-01

    The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit. However, the underlying mechanism has remained unknown. Here, we show that the p53-induced miR-34 microRNA family mediates repression of c-Kit by p53 via a conserved seed-matching sequence in the c-Kit 3'-UTR. Ectopic miR-34a resulted in a decrease in Erk signaling and transformation, which was dependent on the down-regulation of c-Kit expression. Furthermore, ectopic expression of c-Kit conferred resistance of colorectal cancer (CRC) cells to treatment with 5-fluorouracil (5-FU), whereas ectopic miR-34a sensitized the cells to 5-FU. After stimulation with c-Kit ligand/stem cell factor (SCF) Colo320 CRC cells displayed increased migration/invasion, whereas ectopic miR-34a inhibited SCF-induced migration/invasion. Activation of a conditional c-Kit allele induced several stemness markers in DLD-1 CRC cells. In primary CRC samples elevated c-Kit expression also showed a positive correlation with markers of stemness, such as Lgr5, CD44, OLFM4, BMI-1 and β-catenin. On the contrary, activation of a conditional miR-34a allele in DLD-1 cells diminished the expression of c-Kit and several stemness markers (CD44, Lgr5 and BMI-1) and suppressed sphere formation. MiR-34a also suppressed enhanced sphere-formation after exposure to SCF. Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells. Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches.

  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. Does Lin28 Antagonize miRNA-Mediated Repression by Displacing miRISC from Target mRNAs?

    PubMed

    Kallen, Amanda N; Ma, Jing; Huang, Yingqun

    2012-01-01

    Lin28 is a developmentally regulated RNA-binding protein that plays important roles in diverse physiological and pathological processes including oncogenesis and brain synaptic function. These pleiotropic roles of Lin28 are mechanistically linked both to its ability to directly stimulate translation of genes involved primarily in cell growth and metabolism and to its ability to block biogenesis of a subset of miRNAs including the let-7 family of miRNAs. In the case of direct stimulation of gene expression, Lin28 binds to targeted mRNAs through recognition of Lin28-responsive elements (LREs) within mRNAs and recruits RNA helicase A (RHA) to promote translation. RHA belongs to the DEAD-box protein family of RNA helicases, which generally catalyze ATP-dependent unwinding of RNA duplexes or remodeling of ribonucleoprotein complexes (RNPs). Since any given mRNA can potentially be inhibited by miRNAs bearing complementary sequences, we hypothesize that binding of Lin28 to LREs not only nucleates the binding of multiple Lin28 molecules to the same mRNA, but also leads to remodeling of RNPs through recruitment of RHA and causes release of inhibitory miRNA-induced silencing complexes bound to the mRNA. This mode of action may contribute to Lin28-mediated stimulation of translation in both tumor and neuronal cells.

  4. Hepatocellular carcinoma repression by TNFα-mediated synergistic lethal effect of mitosis defect-induced senescence and cell death sensitization.

    PubMed

    Li, Dan; Fu, Jing; Du, Min; Zhang, Haibin; Li, Lu; Cen, Jin; Li, Weiyun; Chen, Xiaotao; Lin, Yunfei; Conway, Edward M; Pikarsky, Eli; Wang, Hongyan; Pan, Guoyu; Ji, Yuan; Wang, Hong-Yang; Hui, Lijian

    2016-10-01

    Hepatocellular carcinoma (HCC) is a cancer lacking effective therapies. Several measures have been proposed to treat HCCs, such as senescence induction, mitotic inhibition, and cell death promotion. However, data from other cancers suggest that single use of these approaches may not be effective. Here, by genetic targeting of Survivin, an inhibitor of apoptosis protein (IAP) that plays dual roles in mitosis and cell survival, we identified a tumor necrosis factor alpha (TNFα)-mediated synergistic lethal effect between senescence and apoptosis sensitization in malignant HCCs. Survivin deficiency results in mitosis defect-associated senescence in HCC cells, which triggers local inflammation and increased TNFα. Survivin inactivation also sensitizes HCC cells to TNFα-triggered cell death, which leads to marked HCC regression. Based on these findings, we designed a combination treatment using mitosis inhibitor and proapoptosis compounds. This treatment recapitulates the therapeutic effect of Survivin deletion and effectively eliminates HCCs, thus representing a potential strategy for HCC therapy. Survivin ablation dramatically suppresses human and mouse HCCs by triggering senescence-associated TNFα and sensitizing HCC cells to TNFα-induced cell death. Combined use of mitotic inhibitor and second mitochondrial-derived activator of caspases mimetic can induce senescence-associated TNFα and enhance TNFα-induced cell death and synergistically eliminate HCC. (Hepatology 2016;64:1105-1120). © 2016 The Authors. (Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.

  5. Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression

    PubMed Central

    Li, Huang; Wang, Zhijia; Zhou, Xuexia; Cheng, Yuanming; Xie, Zhiqin; Manley, James L.; Feng, Ying

    2013-01-01

    Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem–loop structure located downstream of the 3′splice site, and the other involves an exonic splicing silencer (ESS) situated 3′ to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator. PMID:23818605

  6. Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression.

    PubMed

    Li, Huang; Wang, Zhijia; Zhou, Xuexia; Cheng, Yuanming; Xie, Zhiqin; Manley, James L; Feng, Ying

    2013-07-16

    Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem-loop structure located downstream of the 3'splice site, and the other involves an exonic splicing silencer (ESS) situated 3' to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator.

  7. Aspergillus fumigatus spore proteomics and genetics reveal that VeA represses DefA-mediated DNA damage response.

    PubMed

    Shin, Kwang-Soo; Park, Hee-Soo; Kim, Young; Heo, In-Beom; Kim, Young Hwan; Yu, Jae-Hyuk

    2016-10-04

    Aspergillus fumigatus reproduces and infects host by forming a high number of small asexual spores (conidia). The velvet proteins are global transcriptional regulators governing the complex process of conidiogenesis in this fungus. Here, to further understand the velvet-mediated regulation, we carried out comparative proteomic analyses of conidia of wild type (WT) and three velvet mutants (ΔveA, ΔvelB and ΔvosA). Cluster analysis of 184 protein spots showing at least 1.5-fold differential accumulation between WT and mutants reveal the clustering of WT- ΔveA and ΔvelB-ΔvosA. Among 43 proteins identified by Nano-LC-ESI-MS/MS, 23 including several heat shock proteins showed more than two-fold reduction in both the ∆velB and ∆vosA conidia. On the contrary, three proteins exhibited more than five-fold increase in ∆veA only, including the putative RNA polymerase II degradation factor DefA. The deletion of defA resulted in a reduced number of conidia and restricted colony growth. In addition, the defA deletion mutant conidia showed hypersensitivity against the DNA damaging agents NQO and MMS, while the ΔveA mutant conidia were more resistant against to NQO. Taken together, we propose that VeA controls protein level of DefA in conidia, which are dormant and equipped with multiple layers of protection against environmental cues.

  8. Inhibition of COX-2 expression by endocannabinoid 2-arachidonoylglycerol is mediated via PPAR-γ

    PubMed Central

    Du, Huizhi; Chen, Xiaolei; Zhang, Jian; Chen, Chu

    2011-01-01

    BACKGROUND AND PURPOSE Endocannabinoids have both anti-inflammatory and neuroprotective properties against harmful stimuli. We previously demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) protects hippocampal neurons by limiting the inflammatory response via a CB1 receptor-dependent MAPK/NF-κB signalling pathway. The purpose of the present study was to determine whether PPARγ, an important nuclear receptor, mediates 2-AG-induced inhibition of NF-κB phosphorylation and COX-2 expression, and COX-2-enhanced miniature spontaneous excitatory postsynaptic currents (mEPSCs). EXPERIMENTAL APPROACH By using a whole-cell patch clamp electrophysiological recording technique and immunoblot analysis, we determined mEPSCs, expression of COX-2 and PPARγ, and phosphorylation of NF-kB in mouse hippocampal neurons in culture. KEY RESULTS Exogenous and endogenous 2-AG-produced suppressions of NF-κB-p65 phosphorylation, COX-2 expression and excitatory synaptic transmission in response to pro-inflammatory interleukin-1β (IL-1β) and LPS were inhibited by GW9662, a selective PPARγ antagonist, in hippocampal neurons in culture. PPARγ agonists 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and rosiglitazone mimicked the effects of 2-AG on NF-κB-p65 phosphorylation, COX-2 expression and mEPSCs, and these effects were eliminated by antagonism of PPARγ. Moreover, exogenous application of 2-AG or elevation of endogenous 2-AG by inhibiting its hydrolysis with URB602 or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), prevented the IL-1β- and LPS-induced reduction of PPARγ expression. The 2-AG restoration of the reduced PPARγ expression was blocked or attenuated by pharmacological or genetic inhibition of the CB1 receptor. CONCLUSIONS AND IMPLICATIONS Our results suggest that CB1 receptor-dependent PPARγ expression is an important and novel signalling pathway in endocannabinoid 2-AG-produced resolution of neuroinflammation in response to pro

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

    PubMed

    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(+)-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.

  10. EZH2-mediated repression of GSK-3β and TP53 promotes Wnt/β-catenin signaling-dependent cell expansion in cervical carcinoma.

    PubMed

    Chen, Qian; Zheng, Peng-Sheng; Yang, Wen-Ting

    2016-06-14

    Enhancer of zeste homolog 2 (EZH2), a catalytic core component of the Polycomb repressive complex 2 (PRC2), stimulates the silencing of target genes through histone H3 lysine 27 trimethylation (H3K27me3). Recent findings have indicated EZH2 is involved in the development and progression of various human cancers. However, the exact mechanism of EZH2 in the promotion of cervical cancer is largely unknown. Here, we show that EZH2 expression gradually increases during the progression of cervical cancer. We identified a significant positive correlation between EZH2 expression and cell proliferation in vitro and tumor formation in vivo by the up-regulation or down-regulation of EZH2 using CRISPR-Cas9-mediated gene editing technology and shRNA in HeLa and SiHa cells. Further investigation indicated that EZH2 protein significantly accelerated the cell cycle transition from the G0/G1 to S phase. TOP/FOP-Flash reporter assay revealed that EZH2 significantly activated Wnt/β-catenin signaling and the target genes of Wnt/β-catenin pathway were up-regulated, including β-catenin, cyclin D1, and c-myc. Moreover, dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays confirmed that EZH2 inhibited the expression of glycogen synthase kinase-3β (GSK-3β) and TP53 through physically interacting with motifs in the promoters of the GSK-3β and TP53 genes. Additionally, blockage of the Wnt/β-catenin pathway resulted in significant inhibition of cell proliferation, and activation of the Wnt/β-catenin pathway resulted in significant enhancement of cell proliferation, as induced by EZH2. Taken together, our data demonstrate that EZH2 promotes cell proliferation and tumor formation in cervical cancer through activating the Wnt/β-catenin pathway by epigenetic silencing via GSK-3β and TP53.

  11. The Anoikis Effector Bit1 Inhibits EMT through Attenuation of TLE1-Mediated Repression of E-Cadherin in Lung Cancer Cells

    PubMed Central

    Yao, Xin; Pham, Tri; Temple, Brandi; Gray, Selena; Cannon, Cornita; Chen, Renwei; Abdel-Mageed, Asim B.; Biliran, Hector

    2016-01-01

    The mitochondrial Bcl-2 inhibitor of transcription 1 (Bit1) protein is part of an anoikis-regulating pathway that is selectively dependent on integrins. We previously demonstrated that the caspase-independent apoptotic effector Bit1 exerts tumor suppressive function in lung cancer in part by inhibiting anoikis resistance and anchorage-independent growth in vitro and tumorigenicity in vivo. Herein we show a novel function of Bit1 as an inhibitor cell migration and epithelial–mesenchymal transition (EMT) in the human lung adenocarcinoma A549 cell line. Suppression of endogenous Bit1 expression via siRNA and shRNA strategies promoted mesenchymal phenotypes, including enhanced fibroblastoid morphology and cell migratory potential with concomitant downregulation of the epithelial marker E-cadherin expression. Conversely, ectopic Bit1 expression in A549 cells promoted epithelial transition characterized by cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Specific downregulation of E-cadherin in Bit1-transfected cells was sufficient to block Bit1-mediated inhibition of cell motility while forced expression of E-cadherin alone attenuated the enhanced migration of Bit1 knockdown cells, indicating that E-cadherin is a downstream target of Bit1 in regulating cell motility. Furthermore, quantitative real-time PCR and reporter analyses revealed that Bit1 upregulates E-cadherin expression at the transcriptional level through the transcriptional regulator Amino-terminal Enhancer of Split (AES) protein. Importantly, the Bit1/AES pathway induction of E-cadherin expression involves inhibition of the TLE1-mediated repression of E-cadherin, by decreasing TLE1 corepressor occupancy at the E-cadherin promoter as revealed by chromatin immunoprecipitation assays. Consistent with its EMT inhibitory function, exogenous Bit1 expression significantly suppressed the formation of lung metastases of A549 cells in an in vivo experimental

  12. The Anoikis Effector Bit1 Inhibits EMT through Attenuation of TLE1-Mediated Repression of E-Cadherin in Lung Cancer Cells.

    PubMed

    Yao, Xin; Pham, Tri; Temple, Brandi; Gray, Selena; Cannon, Cornita; Chen, Renwei; Abdel-Mageed, Asim B; Biliran, Hector

    The mitochondrial Bcl-2 inhibitor of transcription 1 (Bit1) protein is part of an anoikis-regulating pathway that is selectively dependent on integrins. We previously demonstrated that the caspase-independent apoptotic effector Bit1 exerts tumor suppressive function in lung cancer in part by inhibiting anoikis resistance and anchorage-independent growth in vitro and tumorigenicity in vivo. Herein we show a novel function of Bit1 as an inhibitor cell migration and epithelial-mesenchymal transition (EMT) in the human lung adenocarcinoma A549 cell line. Suppression of endogenous Bit1 expression via siRNA and shRNA strategies promoted mesenchymal phenotypes, including enhanced fibroblastoid morphology and cell migratory potential with concomitant downregulation of the epithelial marker E-cadherin expression. Conversely, ectopic Bit1 expression in A549 cells promoted epithelial transition characterized by cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Specific downregulation of E-cadherin in Bit1-transfected cells was sufficient to block Bit1-mediated inhibition of cell motility while forced expression of E-cadherin alone attenuated the enhanced migration of Bit1 knockdown cells, indicating that E-cadherin is a downstream target of Bit1 in regulating cell motility. Furthermore, quantitative real-time PCR and reporter analyses revealed that Bit1 upregulates E-cadherin expression at the transcriptional level through the transcriptional regulator Amino-terminal Enhancer of Split (AES) protein. Importantly, the Bit1/AES pathway induction of E-cadherin expression involves inhibition of the TLE1-mediated repression of E-cadherin, by decreasing TLE1 corepressor occupancy at the E-cadherin promoter as revealed by chromatin immunoprecipitation assays. Consistent with its EMT inhibitory function, exogenous Bit1 expression significantly suppressed the formation of lung metastases of A549 cells in an in vivo experimental

  13. 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

  14. Bombyx mori E26 transformation-specific 2 (BmEts2), an Ets family protein, represses Bombyx mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in the silkworm Bombyx mori.

    PubMed

    Tanaka, H; Sagisaka, A; Suzuki, N; Yamakawa, M

    2016-10-01

    E26 transformation-specific (Ets) family transcription factors are known to play roles in various biological phenomena, including immunity, in vertebrates. However, the mechanisms by which Ets proteins contribute to immunity in invertebrates remain poorly understood. In this study, we identified a cDNA encoding BmEts2, which is a putative orthologue of Drosophila Yan and human translocation-ets-leukemia/Ets-variant gene 6, from the silkworm Bombyx mori. Expression of the BmEts2 gene was significantly increased in the fat bodies of silkworm larvae in response to injection with Escherichia coli and Staphylococcus aureus. BmEts2 overexpression dramatically repressed B. mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in silkworm cells. Conversely, gene knockdown of BmEts2 significantly enhanced BmRels activity. In addition, two κB sites located on the 5' upstream region of cecropin B1 were found to be involved in the repression of BmRels-mediated promoter activation. Protein-competition analysis further demonstrated that BmEts2 competitively inhibited binding of BmRels to κB sites. Overall, BmEts2 acts as a repressor of BmRels-mediated transactivation of antimicrobial protein genes by inhibiting the binding of BmRels to κB sites. © 2016 The Royal Entomological Society.

  15. Differential selectivity of protein modification by the cyclopentenone prostaglandins PGA1 and 15-deoxy-Delta12,14-PGJ2: role of glutathione.

    PubMed

    Gayarre, Javier; Stamatakis, Konstantinos; Renedo, Marta; Pérez-Sala, Dolores

    2005-10-24

    Cyclopentenone prostaglandins (cyPG) with antiinflammatory and antiproliferative properties have been envisaged as leads for the development of therapeutic agents. Because cyPG effects are mediated in part by the formation of covalent adducts with critical signaling proteins, it is important to assess the specificity of this interaction. By using biotinylated derivatives of 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)-B) and PGA(1) (PGA(1)-B) we herein provide novel evidence for the differential selectivity of protein modification by distinct cyPG. The marked quantitative and qualitative differences in the binding of 15d-PGJ(2)-B and PGA(1)-B to cellular proteins were related to a differential reactivity in the presence of glutathione (GSH), both in vitro and in intact cells. Therefore GSH levels may influence not only the intensity but also the specificity of cyPG action.

  16. 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

  17. 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.

  18. Nrf2 activation protects the liver from ischemia/ reperfusion injury in mice

    PubMed Central

    Kudoh, Kazuhiro; Uchinami, Hiroshi; Yoshioka, Masato; Seki, Ekihiro; Yamamoto, Yuzo

    2014-01-01

    Objective To investigate the role of Nrf2 in the pathogenesis of hepatic ischemia-reperfusion (I/R) injury. Summary Background Data Hepatic I/R injury is a serious complication that leads to liver failure after liver surgery. NF-E2-related factor 2 (Nrf2) is a transcription factor that plays a critical role in protecting cells against oxidative stress. Therefore, it is suggested that Nrf2 activation protects the liver from I/R injury. Methods Wild-type (WT) and Nrf2-deficient mice were treated with 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2), or a vehicle. Subsequently, these mice were subjected to 60 min hepatic 70% ischemia followed by reperfusion. Liver and blood samples were collected to evaluate liver injury and mRNA expressions. Results After hepatic I/R, Nrf2-deficient livers exhibited enhanced tissue damage, impaired GSTm1, NQO1, and GCLc inductions, disturbed redox state, and aggravated TNF-α mRNA expression in comparison to WT livers. 15d-PGJ2 treatment protected the livers of WT mice from I/R injury via increased expressions of GSTm1, NQO1 and GCLc, maintained redox status, and decreased TNF-α induction. These effects induced by 15d-PGJ2 were not seen in the livers of Nrf2−/− mice and were not annulled by PPARγ antagonist in Nrf2+/+ mice, suggesting that the protective effect of 15d-PGJ2 is mediated by Nrf2-dependent antioxidant response. Conclusions Nrf2 plays a critical role in the mechanism of hepatic I/R injury and would be a new therapeutic target for preventing hepatic I/R injury during liver surgery. PMID:24368646

  19. Repression domains of class II ERF transcriptional repressors share an essential motif for active repression.

    PubMed

    Ohta, M; Matsui, K; Hiratsu, K; Shinshi, H; Ohme-Takagi, M

    2001-08-01

    We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif (L)/(F)DLN(L)/(F)(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

  20. 15-deoxy-Δ12,14-PGJ2 induces synoviocyte apoptosis and suppresses adjuvant-induced arthritis in rats

    PubMed Central

    Kawahito, Yutaka; Kondo, Motoharu; Tsubouchi, Yasunori; Hashiramoto, Akira; Bishop-Bailey, David; Inoue, Ken-ichiro; Kohno, Masataka; Yamada, Ryoji; Hla, Timothy; Sano, Hajime

    2000-01-01

    Peroxisome proliferator–activated receptors (PPARs) are members of the nuclear hormone receptor superfamily and have a dominant regulatory role in adipocyte and monocyte differentiation. PPAR-γ agonists are also negative regulators of macrophage activation and have modulatory effects on tumorigenesis. In this study we demonstrate that synovial tissue localized expression of PPAR-γ in patients with rheumatoid arthritis (RA). We detected markedly enhanced expression of PPAR-γ in macrophages, as well as modestly enhanced expression in the synovial lining layer, fibroblasts, and endothelial cells. Activation of the PPAR-γ by 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and the synthetic PPAR-γ ligand (troglitazone) induced RA synoviocyte apoptosis in vitro. Moreover, intraperitoneal administration of these PPAR-γ ligands ameliorated adjuvant-induced arthritis with suppression of pannus formation and mononuclear cell infiltration in female Lewis rats. Anti-inflammatory effects of 15d-PGJ2 were more potent than troglitazone. These findings suggest that PPAR-γ may be an important immunoinflammatory mediator and its ligands, especially 15d-PGJ2, may be useful in the treatment of RA. PMID:10903334

  1. Pseudomonas aeruginosa infection augments inflammation through miR-301b repression of c-Myb-mediated immune activation and infiltration

    PubMed Central

    Li, Xuefeng; He, Sisi; Li, Rongpeng; Zhou, Xikun; Zhang, Shuang; Yu, Min; Ye, Yan; Wang, Yongsheng; Huang, Canhua; Wu, Min

    2016-01-01

    microRNAs (miRNAs) play critical roles in various biological processes including cell proliferation, development, and host defense. However, the molecular mechanism for miRNAs in regulating bacterial-induced inflammation remains largely unclear. Here we report that miR-301b augments pro-inflammatory response during pulmonary infection and caffeine (CAF) suppresses miR-301b’s effect and thereby augmenting respiratory immunity. LPS treatment or Pseudomonas aeruginosa infection induces miR-301b expression via a TLR4/MyD88/NF-κB pathway. Importantly, CAF decreases miR-301b expression through negative regulation of the cAMP/PKA/NF-κB axis. Further, c-Myb is identified as a target of miR-301b, which positively modulates anti-inflammatory cytokines IL-4 and TGF-β1, but negatively regulates pro-inflammatory cytokines MIP-1α and IL-17A. Moreover, repression of miR-301b results in increased transcription of c-Myb and elevated levels of neutrophil infiltration, thereby alleviating infectiou symptoms in mice. These findings reveal miR-301b as a new controller of inflammatory response by repressing c-Myb function to inhibit anti-inflammatory response to bacterial infection, representing a novel mechanism for balancing inflammation. PMID:27670114

  2. 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.

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

    PubMed Central

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

    2016-01-01

    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

  4. The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum

    PubMed Central

    Weber, Barbara; Lindell, Kristoffer; El Qaidi, Samir; Hjerde, Erik; Willassen, Nils-Peder

    2011-01-01

    Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ54 and the σ54-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment. PMID:21948044

  5. RNA polymerase III under control: repression and de-repression.

    PubMed

    Boguta, Magdalena; Graczyk, Damian

    2011-09-01

    The synthesis of tRNA by yeast RNA polymerase III (Pol III) is regulated in response to changing environmental conditions. This control is mediated by Maf1, the global negative regulator of Pol III transcription conserved from yeast to humans. Details regarding the molecular basis of Pol III repression by Maf1 are now emerging from recently reported structural and biochemical data on Pol III and Maf1. Efficient Pol III transcription, following the shift of cells from a non-fermentable carbon source to glucose, requires phosphorylation of Maf1. One of the newly identified Maf1 kinases is the chromatin-bound casein kinase II (CK2). Current studies have allowed us to propose an innovative mechanism of Pol III regulation. We suggest that CK2-mediated phosphorylation of Maf1, occurring directly on tDNA chromatin, controls Pol III recycling. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Searching for repressed memory.

    PubMed

    McNally, Richard J

    2012-01-01

    This chapter summarizes the work of my research group on adults who report either repressed, recovered, or continuous memories of childhood sexual abuse (CSA) or who report no history of CSA. Adapting paradigms from cognitive psychology, we tested hypotheses inspired by both the "repressed memory" and "false memory" perspectives on recovered memories of CSA. We found some evidence for the false memory perspective, but no evidence for the repressed memory perspective. However, our work also suggests a third perspective on recovered memories that does not require the concept of repression. Some children do not understand their CSA when it occurs, and do not experience terror. Years later, they recall the experience, and understanding it as abuse, suffer intense distress. The memory failed to come to mind for years, partly because the child did not encode it as terrifying (i.e., traumatic), not because the person was unable to recall it.

  7. Repression of DOK7 mediated by DNMT3A promotes the proliferation and invasion of KYSE410 and TE-12 ESCC cells.

    PubMed

    Yang, Shou-Mei; Li, Su-Yi; Yu, Hao-Bin; Li, Jie-Ru; Sun, Lei-Lei

    2017-03-23

    Increasing evidence shows that aberrant epigenetic regulation of tumor suppressor genes is a contributing factor to their altered expression in esophageal squamous cell carcinoma (ESCC). In the current study, we investigate the role of DOK7 in ESCC cells. We found that enforced expression of DOK7 inhibited the proliferation and invasion of ESCC cells. We also found that treatment of ESCC cells with the DNA methylation inhibitor, 5-aza-2-deoxycytidine (5-azadC), induced the demethylation of DOK7 in promoter and DOK7 expression. Moreover, silencing DNMT3A decreased methylation of DOK7 and increased DOK7 expression, followed by repressing the proliferation and invasion of ESCC cells. Collectively, our data indicated that silencing DNMT3A inhibits proliferation and invasion in ESCC cells by inducing demethylation of DOK7.

  8. 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. © 2013 AlphaMed Press.

  9. Cohesin protein SMC1 represses the nuclear receptor CAR-mediated synergistic activation of a human P450 gene by xenobiotics.

    PubMed

    Inoue, Kaoru; Borchers, Christoph H; Negishi, Masahiko

    2006-08-15

    CAR (constitutive active/androstane receptor) regulates both the distal enhancer PBREM (phenobarbital-responsive enhancer module) and the proximal element OARE [OA (okadaic acid) response element] to synergistically up-regulate the endogenous CYP2B6 (where CYP is cytochrome P450) gene in HepG2 cells. In this up-regulation, CAR acts as both a transcription factor and a co-regulator, directly binding to and enhancing PBREM upon activation by xenobiotics such as TCPOBOP {1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene} and indirectly associating with the OARE in response to OA [Swales, Kakizaki, Yamamoto, Inoue, Kobayashi and Negishi (2005) J. Biol. Chem. 280, 3458-3466]. We have now identified the cohesin protein SMC1 (structural maintenance of chromosomes 1) as a CAR-binding protein and characterized it as a negative regulator of OARE activity, thus repressing synergy. Treatment with SMC1 small interfering RNA augmented the synergistic up-regulation of CYP2B6 expression 20-fold in HepG2 cells, while transient co-expression of spliced form of SMC1 abrogated the synergistic activation of a 1.8 kb CYP2B6 promoter. SMC1 indirectly binds to a 19 bp sequence (-236/-217) immediately downstream from the OARE in the CYP2B6 promoter. Both DNA affinity and chromatin immunoprecipitation assays showed that OA treatment dissociates SMC1 from the CYP2B6 promoter, reciprocating the indirect binding of CAR to OARE. These results are consistent with the conclusion that SMC1 binding represses OARE activity and its dissociation allows the recruitment of CAR to the OARE, synergizing PBREM activity and the expression of the CYP2B6 gene.

  10. Upregulation of miR21 and Repression of Grhl3 by Leptin Mediates Sinusoidal Endothelial Injury in Experimental Nonalcoholic Steatohepatitis

    PubMed Central

    Pourhoseini, Sahar; Seth, Ratanesh Kumar; Das, Suvarthi; Dattaroy, Diptadip; Kadiiska, Maria B.; Xie, Guanhua; Michelotti, Gregory A.; Nagarkatti, Mitzi; Diehl, Anna Mae; Chatterjee, Saurabh

    2015-01-01

    Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4–5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH. PMID:25658689

  11. Human Sir2-related protein SIRT1 associates with the bHLH repressors HES1 and HEY2 and is involved in HES1- and HEY2-mediated transcriptional repression.

    PubMed

    Takata, Takehiko; Ishikawa, Fuyuki

    2003-01-31

    The Hairy-related bHLH proteins function as transcriptional repressors in most cases and play important roles in diverse aspects of metazoan development. Recently, it was shown that the Drosophila bHLH repressor proteins, Hairy and Deadpan, bind to and function with the NAD(+)-dependent histone deacetylase, Sir2. Here we demonstrate that the human Sir2 homologue, SIRT1, also physically associates with the human bHLH repressor proteins, hHES1 and hHEY2, both in vitro and in vivo. Moreover, using the reporter assay, we show that both SIRT1-dependent and -independent deacetylase pathways are involved in the transcriptional repressions mediated by these bHLH repressors. These results indicate that the molecular association between bHLH proteins and Sir2-related proteins is conserved among metazoans, from Drosophila to human, and suggest that the Sir2-bHLH interaction also plays important roles in human cells.

  12. 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

  13. 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

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

    2015-12-01

    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

  14. 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

  15. H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO.

    PubMed

    Westra, Edze R; Pul, Umit; Heidrich, Nadja; Jore, Matthijs M; Lundgren, Magnus; Stratmann, Thomas; Wurm, Reinhild; Raine, Amanda; Mescher, Melina; Van Heereveld, Luc; Mastop, Marieke; Wagner, E Gerhart H; Schnetz, Karin; Van Der Oost, John; Wagner, Rolf; Brouns, Stan J J

    2010-09-01

    The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.

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

    PubMed

    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.

  17. Z-ligustilide restores tamoxifen sensitivity of ERα negative breast cancer cells by reversing MTA1/IFI16/HDACs complex mediated epigenetic repression of ERα

    PubMed Central

    Dou, Guojun; Wang, Chengqiang; Li, Juan; He, Hui; Wu, Mingxia; Qi, Hongyi

    2017-01-01

    Emerging evidence indicates epigenetic modification represses estrogen receptor α (ERα) and contributes to the resistance to tamoxifen in aggressive ERα-negative (ERα−) breast cancer. Z-ligustilide is a major compound in Radix Angelica sinensis, an herb from traditional Chinese medicine (TCM) most frequently prescribed for breast cancer. However, the role of Z-ligustilide in ERα− breast cancer and epigenetic modification remains largely unknown. Herein we showed, for the first time, that Z-ligustilide restored the growth inhibition of tamoxifen on ERα− breast cancer cells. Apoptosis and S and G2/M phases cell cycle arrest were induced by combinatorial Z-ligustilide and tamoxifen. Importantly, Z-ligustilide reactivated the ERα expression and transcriptional activity, which is proved to be indispensable for restoring the sensitivity to tamoxifen. Interestingly, Z-ligustilide increased Ace-H3 (lys9/14) enrichment in the ERα promoter. Moreover, Z-ligustilide dramatically reduced the enrichment of metastasis-associated protein 1 (MTA1) as well as IFN-γ-inducible protein 16 (IFI16) and histone deacetylases (HDACs) onto the ERα promoter. Meanwhile, Z-ligustilide downregulated MTA1, IFI16 and HDACs, which caused destabilization of the corepressor complex. Collectively, our study not only highlights Z-ligustilide as a novel epigenetic modulator, but also opens new possibilities from TCM for treating aggressive tamoxifen-resistant breast cancer. PMID:28415616

  18. Z-ligustilide restores tamoxifen sensitivity of ERa negative breast cancer cells by reversing MTA1/IFI16/HDACs complex mediated epigenetic repression of ERa.

    PubMed

    Ma, Hui; Li, Li; Dou, Guojun; Wang, Chengqiang; Li, Juan; He, Hui; Wu, Mingxia; Qi, Hongyi

    2017-04-25

    Emerging evidence indicates epigenetic modification represses estrogen receptor α (ERα) and contributes to the resistance to tamoxifen in aggressive ERα-negative (ERα-) breast cancer. Z-ligustilide is a major compound in Radix Angelica sinensis, an herb from traditional Chinese medicine (TCM) most frequently prescribed for breast cancer. However, the role of Z-ligustilide in ERα- breast cancer and epigenetic modification remains largely unknown. Herein we showed, for the first time, that Z-ligustilide restored the growth inhibition of tamoxifen on ERα- breast cancer cells. Apoptosis and S and G2/M phases cell cycle arrest were induced by combinatorial Z-ligustilide and tamoxifen. Importantly, Z-ligustilide reactivated the ERα expression and transcriptional activity, which is proved to be indispensable for restoring the sensitivity to tamoxifen. Interestingly, Z-ligustilide increased Ace-H3 (lys9/14) enrichment in the ERα promoter. Moreover, Z-ligustilide dramatically reduced the enrichment of metastasis-associated protein 1 (MTA1) as well as IFN-γ-inducible protein 16 (IFI16) and histone deacetylases (HDACs) onto the ERα promoter. Meanwhile, Z-ligustilide downregulated MTA1, IFI16 and HDACs, which caused destabilization of the corepressor complex. Collectively, our study not only highlights Z-ligustilide as a novel epigenetic modulator, but also opens new possibilities from TCM for treating aggressive tamoxifen-resistant breast cancer.

  19. miR-155 activates cytokine gene expression in Th17 cells by regulating the DNA-binding protein Jarid2 to relieve polycomb-mediated repression.

    PubMed

    Escobar, Thelma M; Kanellopoulou, Chrysi; Kugler, David G; Kilaru, Gokhul; Nguyen, Cuong K; Nagarajan, Vijayaraj; Bhairavabhotla, Ravikiran K; Northrup, Daniel; Zahr, Rami; Burr, Patrick; Liu, Xiuhuai; Zhao, Keji; Sher, Alan; Jankovic, Dragana; Zhu, Jinfang; Muljo, Stefan A

    2014-06-19

    Specification of the T helper 17 (Th17) cell lineage requires a well-defined set of transcription factors, but how these integrate with posttranscriptional and epigenetic programs to regulate gene expression is poorly understood. Here we found defective Th17 cell cytokine expression in miR-155-deficient CD4+ T cells in vitro and in vivo. Mir155 was bound by Th17 cell transcription factors and was highly expressed during Th17 cell differentiation. miR-155-deficient Th17 and T regulatory (Treg) cells expressed increased amounts of Jarid2, a DNA-binding protein that recruits the Polycomb Repressive Complex 2 (PRC2) to chromatin. PRC2 binding to chromatin and H3K27 histone methylation was increased in miR-155-deficient cells, coinciding with failure to express Il22, Il10, Il9, and Atf3. Defects in Th17 cell cytokine expression and Treg cell homeostasis in the absence of Mir155 could be partially suppressed by Jarid2 deletion. Thus, miR-155 contributes to Th17 cell function by suppressing the inhibitory effects of Jarid2.

  20. miR-155 activates cytokine gene expression in Th17 cells by regulating the DNA-binding protein Jarid2 to relieve Polycomb-mediated repression

    PubMed Central

    Escobar, Thelma M.; Kanellopoulou, Chrysi; Kugler, David G.; Kilaru, Gokhul; Nguyen, Cuong K.; Nagarajan, Vijayaraj; Bhairavabhotla, Ravikiran K.; Northrup, Daniel; Zahr, Rami; Burr, Patrick; Liu, Xiuhuai; Zhao, Keji; Sher, Alan; Jankovic, Dragana; Zhu, Jinfang; Muljo, Stefan A.

    2014-01-01

    Specification of the T helper 17 (Th17) cell lineage requires a well defined set of transcription factors, but how these integrate with post-transcriptional and epigenetic programs to regulate gene expression is poorly understood. Here we found defective Th17 cell cytokine expression in miR-155-deficient CD4+ T cells in vitro and in vivo. Mir155 was bound by Th17 cell transcription factors and was highly expressed during Th17 cell differentiation. miR-155-deficient-Th17 and -T regulatory (Treg) cells expressed increased amounts of Jarid2, a DNA-binding protein that recruits the Polycomb Repressive Complex 2 (PRC2) to chromatin. PRC2 binding to chromatin and H3K27 histone methylation was increased in miR-155-deficient cells, coinciding with failure to express Il22, Il10, Il9 and Atf3. Defects in Th17 cell cytokine expression and Treg cell homeostasis in the absence of Mir155 could be partially suppressed by Jarid2 deletion. Thus, miR-155 contributes to Th17 cell function by suppressing the inhibitory effects of Jarid2. PMID:24856900

  1. Primary root growth in Arabidopsis thaliana is inhibited by the miR159 mediated repression of MYB33, MYB65 and MYB101.

    PubMed

    Xue, Tao; Liu, Zhenhua; Dai, Xuehuan; Xiang, Fengning

    2017-09-01

    Organ growth is a fundamental developmental process basing on cell proliferation and differentiation. The growth of the plant root is sustained by the activity of the root meristem, a process controlled in part by various transcription factors. Here, the miR159 has been identified as a post transcriptional repressor of root growth, on the basis that the mir159ab double mutant developed a larger meristem than did the wild type, and that it formed longer roots. In the mutant, the abundance of MYB33, MYB65 and MYB101 transcript was substantially increased. When MYB33, MYB65 and MYB101 were replaced by the miR159-resistant forms mMYB33, mMYB65 and mMYB101 respectively, the root meristem was similarly enlarged and the growth of the primary root enhanced. MYB65 activity promoted cell division in the root meristem by accelerating the cell cycle. The data suggest that miR159 acts as a key repressor of the primary root's growth, acting through its repression of MYB65 and consequent blocking of the cell cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. 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.

  3. P53-Mediated Repression of the Reprogramming in Cloned Bovine Embryos Through Direct Interaction with HDAC1 and Indirect Interaction with DNMT3A.

    PubMed

    Ma, P J; Zhang, H; Li, R; Wang, Y S; Zhang, Y; Hua, S

    2015-06-01

    P53 is a transcriptional activator, regulating growth arrest, DNA repair and apoptosis. We found that the expression level of P53 and the epigenetic profiles were significantly different in bovine somatic cell nuclear transfer embryos from those in vitro fertilization (IVF) embryos. So we inferred that abnormally expression of P53 might contribute to the incomplete reprogramming. Using bovine foetal fibroblasts, we constructed and screened a highly efficient shRNA vector targeting bovine P53 gene and then reconstituted somatic cell nuclear transfer embryos (RNAi-SCNT). The results indicated that expression levels of P53 were downregulated significantly in RNAi-SCNT embryos, and the blastulation rate and the total number of cell increased significantly. Moreover, methylation levels of CpG islands located 5' region of OCT4, NANOG, H19 and IGF2R in RNAi -SCNT embryos were significantly normalized to that IVF embryos, and the methylation levels of genome DNA, H3K9 and H4K5 acetylation levels were also returned to levels similar to the IVF embryos. Differentially expressed genes were identified by microarray, and 28 transcripts were found to be significantly different (> twofolds) in RNAi-SCNT embryos compared to the control nuclear transfer embryos (SCNT). Among the 28 differentially expressed transcripts, just HDAC1 and DNMT3A were closely associated with the epigenetic modifications. Finally, ChIP further showed that P53 might repress the epigenetic reprogramming by regulating HDAC1 directly and DNMT3A indirectly. These findings offer significant references to further elucidate the mechanism of epigenetic reprogramming in SCNT embryos. © 2015 Blackwell Verlag GmbH.

  4. Neuronal gene repression in Niemann-Pick type C models is mediated by the c-Abl/HDAC2 signaling pathway

    PubMed Central

    Contreras, Pablo S.; Gonzalez-Zuñiga, Marcelo; González-Hódar, Lila; Yáñez, María José; Dulcey, Andrés; Marugan, Juan; Seto, Edward; Alvarez, Alejandra R.; Zanlungo, Silvana

    2016-01-01

    Background Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of free cholesterol in lysosomes. There are currently no effective FDA-approved treatments for NPC, although in the last years the inhibition of Histone Deacetylases (HDACs) has emerged as a potential treatment for this disease. However, the molecular mechanisms that deregulate HDACs activity in NPC disease are unknown. Previously our group had shown that the proapoptotic tyrosine kinase c-Abl signaling is activated in NPC neurons. Here, we demonstrate that c-Abl activity increases HDAC2 levels inducing neuronal gene repression of key synaptic genes in NPC models. Results Our data show that: i) HDAC2 levels and activity are increased in NPC neuronal models and in Npc1-/- mice; ii) inhibition of c-Abl or c-Abl deficiency prevents the increase of HDAC2 protein levels and activity in NPC neuronal models; iii) c-Abl inhibition decreases the levels of HDAC2 tyrosine phosphorylation; iv) treatment with methyl-β-cyclodextrin and Vitamin E decrease the activation of the c-Abl/HDAC2 pathway in NPC neurons; v) in vivo treatment with two c-Abl inhibitors prevents the increase of HDAC2 protein levels in the brain of Npc1-/- mice and, vi) c-Abl inhibition prevents HDAC2 recruitment to the promoter of neuronal genes, triggering an increase in their expression. Conclusion our data show the involvement of the c-Abl/HDAC2 signaling pathway in the regulation of neuronal gene expression in NPC neuronal models. Thus, inhibition of c-Abl could be a pharmacological target for preventing the deleterious effects of increased HDAC2 levels in NPC disease. PMID:26603102

  5. The developmental regulator protein Gon4l associates with protein YY1, co-repressor Sin3a, and histone deacetylase 1 and mediates transcriptional repression.

    PubMed

    Lu, Ping; Hankel, Isaiah L; Hostager, Bruce S; Swartzendruber, Julie A; Friedman, Ann D; Brenton, Janet L; Rothman, Paul B; Colgan, John D

    2011-05-20

    Genetic studies involving zebrafish and mice have demonstrated that the protein Gon4l (Gon4-like) is essential for hematopoiesis. These studies also suggested that Gon4l regulates gene expression during hematopoietic development, yet the biochemical function of Gon4l has not been defined. Here, we describe the identification of factors that interact with Gon4l and may cooperate with this protein to regulate gene expression. As predicted by polypeptide sequence conservation, Gon4l interacted and co-localized with the DNA-binding protein YY1 (Yin Yang 1). Density gradient sedimentation analysis of protein lysates from mouse M12 B cells showed that Gon4l and YY1 co-sediment with the transcriptional co-repressor Sin3a and its functional partner histone deacetylase (HDAC) 1. Consistent with these results, immunoprecipitation studies showed that Gon4l associates with Sin3a, HDAC1, and YY1 as a part of complexes that form in M12 cells. Sequential immunoprecipitation studies demonstrated that Gon4l, YY1, Sin3a, and HDAC1 could all associate as components of a single complex and that a conserved domain spanning the central portion of Gon4l was required for formation of this complex. When targeted to DNA, Gon4l repressed the activity of a nearby promoter, which correlated with the ability to interact with Sin3a and HDAC1. Our data suggest that Sin3a, HDAC1, and YY1 are co-factors for Gon4l and that Gon4l may function as a platform for the assembly of complexes that regulate gene expression.

  6. rAAV8-mediated inhibition of miRNA-21 protects mice against the lethal schistosome infection by repressing both IL-13 and TGF-β1 pathways

    PubMed Central

    Zhang, Dongmei; Su, Qin; Sai, Xue; Bai, Ruipu; Chen, Chao; Luo, Xufeng; Gao, Guangping; Pan, Weiqing

    2015-01-01

    Schistosomiasis is a serious parasitic disease in humans, which can lead to liver fibrosis and death. Accumulating evidence indicated that targeting the deregulated microRNAs could mitigate disease outcomes. Here, we showed that progressive hepatic schistosomiasis caused elevation of miR-21 and efficient and sustained inhibition of miR-21 by using highly hepatic tropic adeno-associated virus serotype 8 (rAAV8) protected mice against the lethal schistosome infection through the attenuation of hepatic fibrosis. We demonstrated an additive role of IL-13 and TGF-β1 in up-regulating the miR-21 expression in the hepatic stellate cells (HSCs) by activation of the SMAD proteins. Further, the down-regulation of miR-21 in the HSCs reversed hepatic fibrosis by enhancing SMAD7 expression, thus repressing TGF-β1/Smad and IL-13/Smad pathways. Conclusion Our study revealed the mechanism of IL-13-mediated schistosomiasis hepatic fibrosis by up-regulation of miR-21 and highlights the potential of rAAV8-mediated miR-21 inhibition as a therapeutic intervention for hepatic fibrotic diseases, such as schistosomiasis. PMID:25546547

  7. 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…

  8. The great repression

    PubMed Central

    Hennig, Bianca P.; Fischer, Tamás

    2013-01-01

    The eukaryotic chromatin structure is essential in correctly defining transcription units. Impairing this structure can activate cryptic promoters, and lead to the accumulation of aberrant RNA transcripts. Here we discuss critical pathways that are responsible for the repression of cryptic transcription and the maintenance of genome integrity. PMID:23665541

  9. 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…

  10. 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

  11. 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

    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.

  12. Alleviation of histone H1-mediated transcriptional repression and chromatin compaction by the acidic activation region in chromosomal protein HMG-14.

    PubMed Central

    Ding, H F; Bustin, M; Hansen, U

    1997-01-01

    Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin structure. Consequently, histone H1 activity must be antagonized in order to convert chromatin to a transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a model system, we now demonstrate that the nonhistone chromosomal protein HMG-14, which is known to preferentially associate with active chromatin, completely alleviates histone H1-mediated inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of HMG-14 are mediated through its acidic, C-terminal region. Strikingly, transcriptional and structural activities of HMG-14 are maintained upon replacement of the C-terminal fragment by acidic regions from either GAL4 or HMG-2. These data support the model that the acidic C terminus of HMG-14 is involved in unfolding higher-order chromatin structure to facilitate transcriptional activation of mammalian genes. PMID:9315642

  13. EZH2 and BCL6 Cooperate to Assemble CBX8-BCOR Complex to Repress Bivalent Promoters, Mediate Germinal Center Formation and Lymphomagenesis.

    PubMed

    Béguelin, Wendy; Teater, Matt; Gearhart, Micah D; Calvo Fernández, María Teresa; Goldstein, Rebecca L; Cárdenas, Mariano G; Hatzi, Katerina; Rosen, Monica; Shen, Hao; Corcoran, Connie M; Hamline, Michelle Y; Gascoyne, Randy D; Levine, Ross L; Abdel-Wahab, Omar; Licht, Jonathan D; Shaknovich, Rita; Elemento, Olivier; Bardwell, Vivian J; Melnick, Ari M

    2016-08-08

    The EZH2 histone methyltransferase mediates the humoral immune response and drives lymphomagenesis through formation of bivalent chromatin domains at critical germinal center (GC) B cell promoters. Herein we show that the actions of EZH2 in driving GC formation and lymphoma precursor lesions require site-specific binding by the BCL6 transcriptional repressor and the presence of a non-canonical PRC1-BCOR-CBX8 complex. The chromodomain protein CBX8 is induced in GC B cells, binds to H3K27me3 at bivalent promoters, and is required for stable association of the complex and the resulting histone modifications. Moreover, oncogenic BCL6 and EZH2 cooperate to accelerate diffuse large B cell lymphoma (DLBCL) development and combinatorial targeting of these repressors results in enhanced anti-lymphoma activity in DLBCLs.

  14. Transcription factor T-bet represses TH17 differentiation by preventing Runx1-mediated activation of the RORγt gene

    PubMed Central

    Lazarevic, Vanja; Chen, Xi; Shim, Jae-Hyuck; Hwang, Eun-Sook; Jang, Eunjung; Bolm, Alexandra N.; Oukka, Mohamed; Kuchroo, Vijay K.; Glimcher, Laurie H.

    2011-01-01

    Overactive TH17 responses are tightly linked to the development of autoimmunity, yet the factors that negatively regulate differentiation of this lineage remain unknown. Here, we report that T-bet suppresses the development of the TH17 cell lineage by inhibiting the transcription of Rorc. T-bet interacts with the transcription factor Runx1 and this interaction blocks Runx1-mediated transactivation of Rorc. T-bet residue Tyr304 is required for T-bet-Runx1 complex formation, for blocking Runx1 activity and for inhibiting the TH17 differentiation program. These data reinforce the concept of master regulators that shape immune responses by simultaneously activating one genetic program while silencing the activity of competing regulators in a common progenitor cell. PMID:21151104

  15. 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. © 2016 Wiley Periodicals, Inc.

  16. 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

  17. RNA interference-mediated repression of MtCCD1 in mycorrhizal roots of Medicago truncatula causes accumulation of C27 apocarotenoids, shedding light on the functional role of CCD1.

    PubMed

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

    2008-11-01

    Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C(40) 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 (C(13) cyclohexenone and C(14) 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 C(27) 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 C(27) apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C(27) 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 (C(27)-->C(14)+C(13)), while the first step (C(40)-->C(27)+C(13)) may be catalyzed by CCD7 and/or CCD4 inside plastids.

  18. The E23 early gene of Drosophila encodes an ecdysone-inducible ATP-binding cassette transporter capable of repressing ecdysone-mediated gene activation

    PubMed Central

    Hock, Tommy; Cottrill, Tracy; Keegan, John; Garza, Dan

    2000-01-01

    At the onset of Drosophila metamorphosis, the steroid hormone 20-OH ecdysone directly induces a small number of early puffs in the polytene chromosomes of the larval salivary gland. Proteins encoded by the early genes corresponding to these transcriptional puffs then regulate the activity of both the early puffs themselves and a much larger set of late puffs. Three of these early genes encode transcription factors that play critical regulatory roles during metamorphosis. Here we report the cloning, DNA sequence, genomic structure, ecdysone inducibility, and temporal expression of an early gene residing in the 23E early puff and denoted E23 (Early gene at 23). In contrast to other early genes, E23 encodes a protein with similarity to ATP-binding cassette transporters. Using heat shock-inducible transgenes, we found that E23 overexpression not only produces phenotypic abnormalities and lethality, but also interferes with ecdysone-mediated gene activation, demonstrating that E23 is capable of modulating the ecdysone response. Our results suggest the existence of a previously unrecognized regulatory mechanism for modulating steroid hormone signaling in Drosophila. PMID:10931948

  19. C1q-Mediated Repression of Human Monocytes Is Regulated by Leukocyte-Associated Ig-Like Receptor 1 (LAIR-1)

    PubMed Central

    Son, Myoungsun; Diamond, Betty

    2014-01-01

    Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by abnormal function of both the innate and the adaptive immune system, leading to a loss of tolerance to self-antigens. Monocytes are a key component of the innate immune system and are efficient producers of multiple cytokines. In SLE, inappropriate activation of monocytes is thought to contribute to the loss of self-tolerance. In this study, we demonstrate that type 1 interferon (IFN) production by CpG-challenged monocytes can be suppressed by C1q through activating leukocyte-associated Ig-like receptor-1 (LAIR-1), which contains immunoreceptor tyrosine-based inhibition motifs (ITIMs). The phosphorylation of LAIR-1 and the interaction of LAIR-1 with SH2 domain–containing protein tyrosine phosphatase-1 (SHP-1) were enhanced after LAIR-1 engagement by C1q. Moreover, engagement of LAIR-1 by C1q inhibited nuclear translocation of interferon regulatory factor (IRF)-3 and IRF5 in CpG-stimulated monocytes. These data suggest a model in which LAIR-1 engagement by C1q helps maintain monocyte tolerance, specifically with respect to Toll-like receptor-9–mediated monocyte activation. PMID:25247291

  20. Nemo phosphorylates Even-skipped and promotes Eve-mediated repression of odd-skipped in even parasegments during Drosophila embryogenesis.

    PubMed

    Braid, Lorena R; Lee, Wendy; Uetrecht, Andrea C; Swarup, Sharan; Papaianni, Gina; Heiler, Amanda; Verheyen, Esther M

    2010-07-01

    Drosophila nemo (nmo) and other Nemo-like kinase family members (Nlks) are well-established key regulators of numerous conserved signaling pathways, such as Wg and BMP. nmo mutants display pleiotropic defects at different developmental stages, including the embryo. In this study we describe a detailed characterization of embryonic cuticle patterning defects associated with maternal loss of nmo. nmo mutant embryos consistently show segmentation defects, most frequently fusions of pairs of denticle belts in alternating segments. These phenotypes are reminiscent of those associated with defects in pair-rule patterning. Genetic interaction studies demonstrate that Nmo promotes Even-skipped (Eve) activity and is required to promote the expression of the Eve target, engrailed (en), in even numbered parasegments. We find that Nmo regulates a subset of Eve activities by stimulating Eve-mediated suppression of the odd-skipped (odd) repressor. Furthermore, we isolate Nmo in a protein complex with Eve and show that Nmo phosphorylates Eve in in vitro kinase assays. These studies reveal a novel role for the Nmo kinase in embryonic pattern formation through its regulation of the homeodomain-containing transcription factor Eve. Copyright 2010 Elsevier Inc. All rights reserved.

  1. Rice osa-miR171c Mediates Phase Change from Vegetative to Reproductive Development and Shoot Apical Meristem Maintenance by Repressing Four OsHAM Transcription Factors.

    PubMed

    Fan, Tian; Li, Xiumei; Yang, Wu; Xia, Kuaifei; Ouyang, Jie; Zhang, Mingyong

    2015-01-01

    Phase change from vegetative to reproductive development is one of the critical developmental steps in plants, and it is regulated by both environmental and endogenous factors. The maintenance of shoot apical meristem (SAM) identity, miRNAs and flowering integrators are involved in this phase change process. Here, we report that the miRNA osa-miR171c targets four GRAS (GAI-RGA-SCR) plant-specific transcription factors (OsHAM1, OsHAM2, OsHAM3, and OsHAM4) to control the floral transition and maintenance of SAM indeterminacy in rice (Oryza sativa). We characterized a rice T-DNA insertion delayed heading (dh) mutant, where the expression of OsMIR171c gene is up-regulated. This mutant showed pleiotropic phenotypic defects, including especially prolonged vegetative phase, delayed heading date, and bigger shoot apex. Parallel expression analysis showed that osa-miR171c controlled the expression change of four OsHAMs in the shoot apex during floral transition, and responded to light. In the dh mutant, the expression of the juvenile-adult phase change negative regulator osa-miR156 was up-regulated, expression of the flowering integrators Hd3a and RFT1 was inhibited, and expression of FON4 negative regulators involved in the maintenance of SAM indeterminacy was also inhibited. From these data, we propose that the inhibition of osa-miR171c-mediated OsHAM transcription factors regulates the phase transition from vegetative to reproductive development by maintaining SAM indeterminacy and inhibiting flowering integrators.

  2. 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

  3. Rice osa-miR171c Mediates Phase Change from Vegetative to Reproductive Development and Shoot Apical Meristem Maintenance by Repressing Four OsHAM Transcription Factors

    PubMed Central

    Yang, Wu; Xia, Kuaifei; Ouyang, Jie; Zhang, Mingyong

    2015-01-01

    Phase change from vegetative to reproductive development is one of the critical developmental steps in plants, and it is regulated by both environmental and endogenous factors. The maintenance of shoot apical meristem (SAM) identity, miRNAs and flowering integrators are involved in this phase change process. Here, we report that the miRNA osa-miR171c targets four GRAS (GAI-RGA-SCR) plant-specific transcription factors (OsHAM1, OsHAM2, OsHAM3, and OsHAM4) to control the floral transition and maintenance of SAM indeterminacy in rice (Oryza sativa). We characterized a rice T-DNA insertion delayed heading (dh) mutant, where the expression of OsMIR171c gene is up-regulated. This mutant showed pleiotropic phenotypic defects, including especially prolonged vegetative phase, delayed heading date, and bigger shoot apex. Parallel expression analysis showed that osa-miR171c controlled the expression change of four OsHAMs in the shoot apex during floral transition, and responded to light. In the dh mutant, the expression of the juvenile-adult phase change negative regulator osa-miR156 was up-regulated, expression of the flowering integrators Hd3a and RFT1 was inhibited, and expression of FON4 negative regulators involved in the maintenance of SAM indeterminacy was also inhibited. From these data, we propose that the inhibition of osa-miR171c-mediated OsHAM transcription factors regulates the phase transition from vegetative to reproductive development by maintaining SAM indeterminacy and inhibiting flowering integrators. PMID:26023934

  4. 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.

  5. The repressed and implicit knowledge.

    PubMed

    Talvitie, Vesa; Ihanus, Juhani

    2002-12-01

    The distinction between implicit (non-conscious) and explicit (conscious) knowledge made by cognitive scientists is applied to the psychoanalytic idea of repressed contents. The consequences of repression are suggested to have been caused by implicit representations. Repressed memories can also be treated in terms of explicit representations, which are prevented from becoming activated. Implicit knowledge cannot, however, be made conscious, and thus the idea of becoming conscious of the repressed desires and fears that have never been conscious is contradictory. This tension may be relieved by reconceptualising the idea of becoming conscious of the repressed. It is suggested that this could be seen as creating explicit knowledge about the effects of implicit representations. By applying the implicit/explicit knowledge distinction, psychoanalytic ideas concerning the repressed could be connected to current views in the domain of cognitive orientation.

  6. 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.

  7. PPARα Promotes Cancer Cell Glut1 Transcription Repression.

    PubMed

    You, Mengli; Jin, Jianhua; Liu, Qian; Xu, QingGang; Shi, Juanjuan; Hou, Yongzhong

    2017-06-01

    Abundant nutrient availability including glucose and amino acids plays an important role in maintaining cancer cell energetic and biosynthetic pathways. As a nuclear receptor, peroxisome-proliferator-activated receptor α (PPARα) regulates inflammation and cancer progression, however, it is still unclear the interaction of PPARα with the cancer cell glucose metabolism. Here we found that PPARα reduced Glut1 (Glucose transporter 1) protein and gene levels in HCT-116, SW480, HeLa, and MCF-7 cancer cell lines. In contrast, silenced PPARα reversed this event. Further analysis shows that PPARα directly targeted the consensus PPRE motif of Glut1 promoter region resulting in Glut1 transcription repression. PPARα-mediated Glut1 transcription repression led to decreased influx of glucose in cancer cells. These findings revealed a novel mechanism of PPARα-mediated cancer cell Glut1 transcription repression. J. Cell. Biochem. 118: 1556-1562, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Immunopathogenesis of HIV infection in cocaine users: role of arachidonic acid.

    PubMed

    Samikkannu, Thangavel; Rao, Kurapati V K; Ding, Hong; Agudelo, Marisela; Raymond, Andrea D; Yoo, Changwon; Nair, Madhavan P N

    2014-01-01

    Arachidonic acid (AA) is known to be increased in HIV infected patients and illicit drug users are linked with severity of viral replication, disease progression, and impaired immune functions. Studies have shown that cocaine accelerates HIV infection and disease progression mediated by immune cells. Dendritic cells (DC) are the first line of antigen presentation and defense against immune dysfunction. However, the role of cocaine use in HIV associated acceleration of AA secretion and its metabolites on immature dendritic cells (IDC) has not been elucidated yet. The aim of this study is to elucidate the mechanism of AA metabolites cyclooxygenase-2 (COX-2), prostaglandin E2 synthetase (PGE2), thromboxane A2 receptor (TBXA2R), cyclopentenone prostaglandins (CyPG), such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), 14-3-3 ζ/δ and 5-lipoxygenase (5-LOX) mediated induction of IDC immune dysfunctions in cocaine using HIV positive patients. The plasma levels of AA, PGE2, 15d-PGJ2, 14-3-3 ζ/δ and IDC intracellular COX-2 and 5-LOX expression were assessed in cocaine users, HIV positive patients, HIV positive cocaine users and normal subjects. Results showed that plasma concentration levels of AA, PGE2 and COX-2, TBXA2R and 5-LOX in IDCs of HIV positive cocaine users were significantly higher whereas 15d-PGJ2 and 14-3-3 ζ/δ were significantly reduced compared to either HIV positive subjects or cocaine users alone. This report demonstrates that AA metabolites are capable of mediating the accelerative effects of cocaine on HIV infection and disease progression.

  9. SIRT1 activation enhances HDAC inhibition-mediated upregulation of GADD45G by repressing the binding of NF-κB/STAT3 complex to its promoter in malignant lymphoid cells

    PubMed Central

    Scuto, A; Kirschbaum, M; Buettner, R; Kujawski, M; Cermak, J M; Atadja, P; Jove, R

    2013-01-01

    We explored the activity of SIRT1 activators (SRT501 and SRT2183) alone and in combination with panobinostat in a panel of malignant lymphoid cell lines in terms of biological and gene expression responses. SRT501 and SRT2183 induced growth arrest and apoptosis, concomitant with deacetylation of STAT3 and NF-κB, and reduction of c-Myc protein levels. PCR arrays revealed that SRT2183 leads to increased mRNA levels of pro-apoptosis and DNA-damage-response genes, accompanied by accumulation of phospho-H2A.X levels. Next, ChIP assays revealed that SRT2183 reduces the DNA-binding activity of both NF-κB and STAT3 to the promoter of GADD45G, which is one of the most upregulated genes following SRT2183 treatment. Combination of SRT2183 with panobinostat enhanced the anti-growth and anti-survival effects mediated by either compound alone. Quantitative-PCR confirmed that the panobinostat in combination with SRT2183, SRT501 or resveratrol leads to greater upregulation of GADD45G than any of the single agents. Panobinostat plus SRT2183 in combination showed greater inhibition of c-Myc protein levels and phosphorylation of H2A.X, and increased acetylation of p53. Furthermore, EMSA revealed that NF-κB binds directly to the GADD45G promoter, while STAT3 binds indirectly in complexes with NF-κB. In addition, the binding of NF-κB/STAT3 complexes to the GADD45G promoter is inhibited following panobinostat, SRT501 or resveratrol treatment. Moreover, the combination of panobinostat with SRT2183, SRT501 or resveratrol induces a greater binding repression than either agent alone. These data suggest that STAT3 is a corepressor with NF-κB of the GADD45G gene and provides in vitro proof-of-concept for the combination of HDACi with SIRT1 activators as a potential new therapeutic strategy in lymphoid malignancies. PMID:23681230

  10. Chromatin Landscape Defined by Repressive Histone Methylation during Oligodendrocyte Differentiation

    PubMed Central

    Liu, Jia; Magri, Laura; Zhang, Fan; Marsh, Nidaa O.; Albrecht, Stefanie; Huynh, Jimmy L.; Kaur, Jasbir; Kuhlmann, Tanja; Zhang, Weijia; Slesinger, Paul A.

    2015-01-01

    In many cell types, differentiation requires an interplay between extrinsic signals and transcriptional changes mediated by repressive and activating histone modifications. Oligodendrocyte progenitors (OPCs) are electrically responsive cells receiving synaptic input. The differentiation of these cells into myelinating oligodendrocytes is characterized by temporal waves of gene repression followed by activation of myelin genes and progressive decline of electrical responsiveness. In this study, we used chromatin isolated from rat OPCs and immature oligodendrocytes, to characterize the genome-wide distribution of the repressive histone marks, H3K9me3 and H3K27me3, during differentiation. Although both marks were present at the OPC stage, only H3K9me3 marks (but not H3K27me3) were found to be increased during differentiation, at genes related to neuronal lineage and regulation of membrane excitability. Consistent with these findings, the levels and activity of H3K9 methyltransferases (H3K9 HMT), but not H3K27 HMT, increased more prominently upon exposure to oligodendrocyte differentiating stimuli and were detected in stage-specific repressive protein complexes containing the transcription factors SOX10 or YY1. Silencing H3K9 HMT, but not H3K27 HMT, impaired oligodendrocyte differentiation and functionally altered the response of oligodendrocytes to electrical stimulation. Together, these results identify repressive H3K9 methylation as critical for gene repression during oligodendrocyte differentiation. PMID:25568127

  11. 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 ...

  12. How social media matter: Repression and the diffusion of the Occupy Wall Street movement.

    PubMed

    Suh, Chan S; Vasi, Ion Bogdan; Chang, Paul Y

    2017-07-01

    This study explores the role played by social media in reshaping the repression-mobilization relationship. Drawing on the case of the Occupy Wall Street movement, we examine the impact of Facebook and Twitter on the spatial diffusion of protests during a period of heightened state repression. Results from event history analyses suggest that the effects of repression on protest diffusion are contingent on the presence of social media accounts supporting the movement. We find that state repression at earlier protest sites encouraged activists to create Facebook and Twitter accounts in their own cities, which then served as important vehicles for the initiation of new Occupy protests. Moreover, results suggest that repression incidents can directly facilitate future protests in cities that already have Occupy Facebook accounts. This study highlights the potential of social media to both mediate and moderate the influence of repression on the diffusion of contemporary movements. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Localization of 14-3-3δ/ξ on the neuronal cell surface.

    PubMed

    Yamamoto, Yasuhiro; Koma, Hiromi; Yagami, Tatsurou

    2015-11-01

    14-3-3 proteins are intracellularly expressed as ubiquitous adaptor proteins. Here, we found localization of 14-3-3δ/ξ on the neuronal cell surface. 14-3-3δ/ξ was identified as a membrane target for 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2). 15d-PGJ2 is a pathological mediator of neurodegenerative diseases including Alzheimer's disease (AD). A causative peptide for AD, amyloid β, is one of binding partner of 14-3-3δ/ξ. Non-permeabilized neurons were used to avoid the intracellular effects of anti-14-3-3δ/ξ antibody in the present study. The plasmalemmal 14-3-3δ/ξ, but not the cytosolic one, was stimulated by its specific antibody, resulting in neuronal cell death. The neurotoxicity of anti-14-3-3δ/ξ antibody was suppressed by an antioxidant, catalase. Catalase prevented neurons from anti-14-3-3δ/ξ antibody-generating neurotoxic H2O2. The neuroprotective effect of catalase was also detected with the post-treatment of neurons after the application of anti-14-3-3δ/ξ antibody. Activation of mitogen-activated protein kinase signaling cascade is a down-stream consequence of H2O2 exposure. A c-Jun N-terminal kinase inhibitor suppressed anti-14-3-3δ/ξ antibody-induced neuronal cell death. To my knowledge, this is the first report that the antibody-stimulated plasmalemmal 14-3-3δ/ξ induced neuronal cell death. Furthermore, H2O2 and JNK contributed to the neurotoxicity of anti-14-3-3δ/ξ antibody as well as those of amyloid β and 15d-PGJ2. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Peroxisome proliferator-activated receptor gamma in human breast carcinoma: a modulator of estrogenic actions.

    PubMed

    Suzuki, T; Hayashi, S; Miki, Y; Nakamura, Y; Moriya, T; Sugawara, A; Ishida, T; Ohuchi, N; Sasano, H

    2006-03-01

    It has been reported that agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibit proliferation of breast carcinoma cells, but the biological significance of PPARgamma remains undetermined in human breast carcinomas. Therefore, we immunolocalized PPARgamma in 238 human breast carcinoma tissues. PPARgamma immunoreactivity was detected in 42% of carcinomas, and was significantly associated with the status of estrogen receptor (ER) alpha, ERbeta, progesterone receptor, retinoic X receptors, p21 or p27, and negatively correlated with histological grade or cyclooxygenase-2 status. PPARgamma immunoreactivity was significantly associated with an improved clinical outcome of breast carcinoma patients by univariate analysis, and multivariate analysis demonstrated that PPARgamma immunoreactivity was an independent prognostic factor for overall survival in ERalpha-positive patients. We then examined possible mechanisms of modulation by PPARgamma on estrogenic actions in MCF-7 breast carcinoma cells. A PPARgamma activator, 15-deoxy-Delta(12,14)- prostaglandin J(2) (15d-PGJ(2)), significantly inhibited estrogen-responsive element-dependent transactivation by estradiol in MCF-7 cells, which was blocked by addition of a PPARgamma antagonist GW9662. Subsequent study, employing a custom-made microarray focused on estrogen-responsive genes, revealed that mRNA expression was significantly regulated by estradiol in 49 genes, but this significance vanished on addition of 15d-PGJ(2) in 16 out of 49 (33%) genes. These findings were confirmed by real-time PCR in 11 genes. 15d-PGJ(2) significantly inhibited estrogen-mediated proliferation of MCF-7 cells, and caused accumulation of p21 and p27 protein. These results suggest that PPARgamma is mainly expressed in well-differentiated and ER-positive breast carcinomas, and modulates estrogenic actions.

  15. The human clotting factor VIII cDNA contains an autonomously replicating sequence consensus- and matrix attachment region-like sequence that binds a nuclear factor, represses heterologous gene expression, and mediates the transcriptional effects of sodium butyrate.

    PubMed Central

    Fallaux, F J; Hoeben, R C; Cramer, S J; van den Wollenberg, D J; Briët, E; van Ormondt, H; van Der Eb, A J

    1996-01-01

    Expression of the human blood-clotting factor VIII (FVIII) cDNA is hampered by the presence of sequences located in the coding region that repress transcription. We have previously identified a 305-bp fragment within the FVIII cDNA that is involved in the repression (R.C. Hoeben, F.J. Fallaux, S.J. Cramer, D.J.M. van den Wollenberg, H. van Ormondt, E. Briet, and A.J. van der Eb, Blood 85:2447-2454, 1995). Here, we show that this 305-bp region of FVIII cDNA contains sequences that resemble the yeast (Saccharomyces cerevisiae) autonomously replicating sequence consensus. Two of these DNA elements coincide with AT-rich sequences that are often found in matrix attachment regions or scaffold-attached regions. One of these elements, consisting of nucleotides 1569 to 1600 of the FVIII cDNA (nucleotide numbering is according to the system of Wood et al. (W.I. Wood, D.J. Capon, C.C. Simonsen, D.L. Eaton, J. Gitschier, D. Keyt, P.H. Seeburg, D.H. Smith, P. Hollingshead, K.L. Wion, et al., Nature [London] 312:330-337,1984), binds a nuclear factor in vitro but loses this capacity after four of its base pairs have been changed. A synthetic heptamer of this segment can repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene and also loses this capacity upon mutation. Furthermore, we demonstrate that repression by FVIII sequences can be relieved by sodium butyrate. We demonstrate that the synthetic heptamer (FVIII nucleotides 1569 to 1600), when placed upstream of the Moloney murine leukemia virus long terminal repeat promoter that drives the CAT reporter, can render the CAT reporter inducible by butyrate. This effect was absent when the same element was mutated. The stimulatory effect of butyrate could not be attributed to butyrate-responsive elements in the studied long terminal repeat promoters. Our data provide a functional characterization of the sequences that repress expression of the FVIII cDNA. These data also suggest a link between

  16. Translational Repression in Malaria Sporozoites

    PubMed Central

    Turque, Oliver; Tsao, Tiffany; Li, Thomas; Zhang, Min

    2016-01-01

    Malaria is a mosquito-borne infectious disease of humans and other animals. It is caused by the parasitic protozoan, Plasmodium. Sporozoites, the infectious form of malaria parasites, are quiescent when they remain in the salivary glands of the Anopheles mosquito until transmission into a mammalian host. Metamorphosis of the dormant sporozoite to its active form in the liver stage requires transcriptional and translational regulations. Here, we summarize recent advances in the translational repression of gene expression in the malaria sporozoite. In sporozoites, many mRNAs that are required for liver stage development are translationally repressed. Phosphorylation of eukaryotic Initiation Factor 2α (eIF2α) leads to a global translational repression in sporozoites. The eIF2α kinase, known as Upregulated in Infectious Sporozoite 1 (UIS1), is dominant in the sporozoite. The eIF2α phosphatase, UIS2, is translationally repressed by the Pumilio protein Puf2. This translational repression is alleviated when sporozoites are delivered into the mammalian host.

  17. 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

  18. Complex regulation of the global regulatory gene csrA: CsrA-mediated translational repression, transcription from five promoters by Eσ⁷⁰ and Eσ(S), and indirect transcriptional activation by CsrA.

    PubMed

    Yakhnin, Helen; Yakhnin, Alexander V; Baker, Carol S; Sineva, Elena; Berezin, Igor; Romeo, Tony; Babitzke, Paul

    2011-08-01

    CsrA of Escherichia coli is an RNA-binding protein that globally regulates gene expression by repressing translation and/or altering the stability of target transcripts. Here we explored mechanisms that control csrA expression. Four CsrA binding sites were predicted upstream of the csrA initiation codon, one of which overlapped its Shine-Dalgarno sequence. Results from gel shift, footprint, toeprint and in vitro translation experiments indicate that CsrA binds to these four sites and represses its own translation by directly competing with 30S ribosomal subunit binding. Experiments were also performed to examine transcription of csrA. Primer extension, in vitro transcription and in vivo expression studies identified two σ⁷⁰-dependent (P2 and P5) and two σ(S) -dependent (P1 and P3) promoters that drive transcription of csrA. Additional primer extension studies identified a fifth csrA promoter (P4). Transcription from P3, which is indirectly activated by CsrA, is primarily responsible for increased csrA expression as cells transition from exponential to stationary-phase growth. Taken together, our results indicate that regulation of csrA expression occurs by a variety of mechanisms, including transcription from multiple promoters by two sigma factors, indirect activation of its own transcription, as well as direct repression of its own translation. © 2011 Blackwell Publishing Ltd.

  19. Complex regulation of the global regulatory gene csrA: CsrA-mediated translational repression, transcription from five promoters by Eσ70 and EσS, and indirect transcriptional activation by CsrA

    PubMed Central

    Yakhnin, Helen; Yakhnin, Alexander V.; Baker, Carol S.; Sineva, Elena; Berezin, Igor; Romeo, Tony; Babitzke, Paul

    2011-01-01

    Summary CsrA of Escherichia coli is an RNA binding protein that globally regulates gene expression by repressing translation and/or altering the stability of target transcripts. Here we explored mechanisms that control csrA expression. Four CsrA binding sites were predicted upstream of the csrA initiation codon, one of which overlapped its Shine-Dalgarno sequence. Results from gel shift, footprint, toeprint and in vitro translation experiments indicate that CsrA binds to these four sites and represses its own translation by directly competing with 30S ribosomal subunit binding. Experiments were also performed to examine transcription of csrA. Primer extension, in vitro transcription and in vivo expression studies identified two σ70-dependent (P2 and P5) and two σS-dependent (P1 and P3) promoters that drive transcription of csrA. Additional primer extension studies identified a fifth csrA promoter (P4). Transcription from P3, which is indirectly activated by CsrA, is primarily responsible for increased csrA expression as cells transition from exponential to stationary phase growth. Taken together, our results indicate that regulation of csrA expression occurs by a variety of mechanisms, including transcription from multiple promoters by two sigma factors, indirect activation of its own transcription, as well as direct repression of its own translation. PMID:21696456

  20. 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.

  1. ATRX represses alternative lengthening of telomeres

    PubMed Central

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

    2015-01-01

    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

  2. Repression of gene expression by oxidative stress.

    PubMed Central

    Morel, Y; Barouki, R

    1999-01-01

    Gene expression is modulated by both physiological signals (hormones, cytokines, etc.) and environmental stimuli (physical parameters, xenobiotics, etc.). Oxidative stress appears to be a key pleiotropic modulator which may be involved in either pathway. Indeed, reactive oxygen species (ROS) have been described as second messengers for several growth factors and cytokines, but have also been shown to rise following cellular insults such as xenobiotic metabolism or enzymic deficiency. Extensive studies on the induction of stress-response genes by oxidative stress have been reported. In contrast, owing to the historical focus on gene induction, less attention has been paid to gene repression by ROS. However, a growing number of studies have shown that moderate (i.e. non-cytotoxic) oxidative stress specifically down-regulates the expression of various genes. In this review, we describe the alteration of several physiological functions resulting from oxidative-stress-mediated inhibition of gene transcription. We will then focus on the repressive oxidative modulation of various transcription factors elicited by ROS. PMID:10477257

  3. 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)

  4. Repression of the Low Affinity Iron Transporter Gene FET4

    PubMed Central

    Caetano, Soraia M.; Menezes, Regina; Amaral, Catarina; Rodrigues-Pousada, Claudina; Pimentel, Catarina

    2015-01-01

    Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, causing several cellular damages and eventually leading to death. In the yeast Saccharomyces cerevisiae, the transcription factor Yap1 plays a key role in the regulation of several genes involved in metal stress response. We have previously shown that Yap1 represses the expression of FET4, a gene encoding a low affinity iron transporter able to transport metals other than iron. Here, we have studied the relevance of this repression in cell tolerance to cadmium. Our results indicate that genomic deletion of Yap1 increases FET4 transcript and protein levels. In addition, the cadmium toxicity exhibited by this strain is completely reversed by co-deletion of FET4 gene. These data correlate well with the increased intracellular levels of cadmium observed in the mutant yap1. Rox1, a well known aerobic repressor of hypoxic genes, conveys the Yap1-mediated repression of FET4. We further show that, in a scenario where the activity of Yap1 or Rox1 is compromised, cells activate post-transcriptional mechanisms, involving the exoribonuclease Xrn1, to compensate the derepression of FET4. Our data thus reveal a novel protection mechanism against cadmium toxicity mediated by Yap1 that relies on the aerobic repression of FET4 and results in the impairment of cadmium uptake. PMID:26063801

  5. 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

  6. Nitric oxide participates in plant flowering repression by ascorbate

    PubMed Central

    Senthil Kumar, Rajendran; Shen, Chin-Hui; Wu, Pei-Yin; Suresh Kumar, Subbiah; Hua, Moda Sang; Yeh, Kai-Wun

    2016-01-01

    In Oncidium, redox homeostasis involved in flowering is mainly due to ascorbic acid (AsA). Here, we discovered that Oncidium floral repression is caused by an increase in AsA-mediated NO levels, which is directed by the enzymatic activities of nitrate reductase (NaR) and nitrite reducatase (NiR). Through Solexa transcriptomic analysis of two libraries, ‘pseudobulb with inflorescent bud’ (PIB) and ‘pseudobulb with axillary bud’ (PAB), we identified differentially expressed genes related to NO metabolism. Subsequently, we showed a significant reduction of NaR enzymatic activities and NO levels during bolting and blooming stage, suggesting that NO controlled the phase transition and flowering process. Applying AsA to Oncidium PLB (protocorm-like bodies) significantly elevated the NO content and enzyme activities. Application of sodium nitroprusside (-NO donor) on Arabidopsis vtc1 mutant caused late flowering and expression level of flowering-associated genes (CO, FT and LFY) were reduced, suggesting NO signaling is vital for flowering repression. Conversely, the flowering time of noa1, an Arabidopsis NO-deficient mutant, was not altered after treatment with L-galacturonate, a precursor of AsA, suggesting AsA is required for NO-biosynthesis involved in the NO-mediated flowering-repression pathway. Altogether, Oncidium bolting is tightly regulated by AsA-mediated NO level and downregulation of transcriptional levels of NO metabolism genes. PMID:27731387

  7. Spinal TLR4 mediates the transition to a persistent mechanical hypersensitivity after the resolution of inflammation in serum-transferred arthritis

    PubMed Central

    Christianson, Christina A.; Dumlao, Darren S.; Stokes, Jennifer A.; Dennis, Edward A.; Svensson, Camilla I.; Corr, Maripat; Yaksh, Tony L.

    2012-01-01

    Persistent pain after resolution of clinically appreciable signs of arthritis poses a therapeutic challenge and immunosuppressive therapies do not meet this medical need. To investigate this conversion to persistent pain, we utilized the K/BxN serum transfer arthritis model, which has persistent mechanical hypersensitivity despite the resolution of visible inflammation. Toll-like receptor (TLR) 4 has been implicated as a potential therapeutic target in neuropathic and other pain models. We compared the relative courses of serum transfer arthritis and mechanical hypersensitivity in wild type (WT) and Tlr4−/− mice. K/BxN serum transfer induced similar joint swelling and inflammation from days 4–22 in WT and Tlr4−/− mice. Unlike WT mice, Tlr4−/− mice displayed a significant reversal in mechanical hypersensitivity and diminished appearance of glial activation markers after resolution of peripheral inflammation. Intrathecal (IT) delivery of a TLR4 antagonist, LPS-RS (10μg), on days 6, 9, and 12 abrogated the transition to persistent mechanical hypersensitivity in WT arthritic mice, while later administration had no impact. We utilized a lipodomics LC/MS/MS methodology to determine spinal cord profiles of bioactive lipid species following early LPS-RS treatment compared to vehicle treated controls. WT arthritic mice had reduced spinal levels of the anti-inflammatory prostaglandin 15d-PGJ2 on day 6, compared to IT LPS-RS treated mice. Direct IT application of 15d-PGJ2 (0.5μg) on day 6 improved mechanical hypersensitivity in arthritic mice within 15 minutes. Hence, TLR4 signaling altered spinal bioactive lipid profiles in the serum transfer model and played a critical role in the transition from acute to chronic post-inflammatory mechanical hypersensitivity. PMID:22019135

  8. MyoR Modulates Cardiac Conduction by Repressing Gata4

    PubMed Central

    Harris, John P.; Bhakta, Minoti; Bezprozvannaya, Svetlana; Wang, Lin; Lubczyk, Christina; Olson, Eric N.

    2014-01-01

    The cardiac conduction system coordinates electrical activation through a series of interconnected structures, including the atrioventricular node (AVN), the central connection point that delays impulse propagation to optimize cardiac performance. Although recent studies have uncovered important molecular details of AVN formation, relatively little is known about the transcriptional mechanisms that regulate AV delay, the primary function of the mature AVN. We identify here MyoR as a novel transcription factor expressed in Cx30.2+ cells of the AVN. We show that MyoR specifically inhibits a Cx30.2 enhancer required for AVN-specific gene expression. Furthermore, we demonstrate that MyoR interacts directly with Gata4 to mediate transcriptional repression. Our studies reveal that MyoR contains two nonequivalent repression domains. While the MyoR C-terminal repression domain inhibits transcription in a context-dependent manner, the N-terminal repression domain can function in a heterologous context to convert the Hand2 activator into a repressor. In addition, we show that genetic deletion of MyoR in mice increases Cx30.2 expression by 50% and prolongs AV delay by 13%. Taken together, we conclude that MyoR modulates a Gata4-dependent regulatory circuit that establishes proper AV delay, and these findings may have wider implications for the variability of cardiac rhythm observed in the general population. PMID:25487574

  9. Repression of vascular endothelial growth factor A in glioblastoma cells using engineered zinc finger transcription factors.

    PubMed

    Snowden, Andrew W; Zhang, Lei; Urnov, Fyodor; Dent, Carolyn; Jouvenot, Yann; Zhong, Xiaohong; Rebar, Edward J; Jamieson, Andrew C; Zhang, H Steven; Tan, Siyuan; Case, Casey C; Pabo, Carl O; Wolffe, Alan P; Gregory, Philip D

    2003-12-15

    Angiogenic factors are necessary for tumor proliferation and thus are attractive therapeutic targets. In this study, we have used engineered zinc finger protein (ZFP) transcription factors (TFs) to repress expression of vascular endothelial growth factor (VEGF)-A in human cancer cell lines. We create potent transcriptional repressors by fusing a designed ZFP targeted to the VEGF-A promoter with either the ligand-binding domain of thyroid hormone receptor alpha or its viral relative, vErbA. Moreover, this ZFP-vErbA repressor binds its intended target site in vivo and mediates the specific deacetylation of histones H3 and H4 at the targeted promoter, a result that emulates the natural repression mechanism of these domains. The potential therapeutic relevance of ZFP-mediated VEGF-A repression was addressed using the highly tumorigenic glioblastoma cell line U87MG. Despite the aberrant overexpression of VEGF-A in this cell line, engineered ZFP TFs were able to repress the expression of VEGF-A by >20-fold. The VEGF-A levels observed after ZFP TF-mediated repression were comparable to those of a nonangiogenic cancer line (U251MG), suggesting that the degree of repression obtained with the ZFP TF would be sufficient to suppress tumor angiogenesis. Thus, engineered ZFP TFs are shown to be potent regulators of gene expression with therapeutic promise in the treatment of disease.

  10. PPARgamma agonists inhibit TGF-beta induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis.

    PubMed

    Burgess, Heather A; Daugherty, Louis Eugene; Thatcher, Thomas H; Lakatos, Heather F; Ray, Denise M; Redonnet, Michelle; Phipps, Richard P; Sime, Patricia J

    2005-06-01

    Pulmonary fibrosis is a progressive life-threatening disease for which no effective therapy exists. Myofibroblasts are one of the key effector cells in pulmonary fibrosis and are the primary source of extracellular matrix production. Drugs that inhibit the differentiation of fibroblasts to myofibroblasts have potential as antifibrotic therapies. Peroxisome proliferator-activated receptor (PPAR)-gamma is a transcription factor that upon ligation with PPARgamma agonists activates target genes containing PPAR response elements. PPARgamma agonists have anti-inflammatory activities and may have potential as antifibrotic agents. In this study, we examined the abilities of PPARgamma agonists to block two of the most important profibrotic activities of TGF-beta on pulmonary fibroblasts: myofibroblast differentiation and production of excess collagen. Both natural (15d-PGJ2) and synthetic (ciglitazone and rosiglitazone) PPARgamma agonists inhibited TGF-beta-driven myofibroblast differentiation, as determined by alpha-smooth muscle actin-specific immunocytochemistry and Western blot analysis. PPARgamma agonists also potently attenuated TGF-beta-driven type I collagen protein production. A dominant-negative PPARgamma partially reversed the inhibition of myofibroblast differentiation by 15d-PGJ2 and rosiglitazone, but the irreversible PPARgamma antagonist GW-9662 did not, suggesting that the antifibrotic effects of the PPARgamma agonists are mediated through both PPARgamma-dependent and independent mechanisms. Thus PPARgamma agonists have novel and potent antifibrotic effects in human lung fibroblasts and may have potential for therapy of fibrotic diseases in the lung and other tissues.

  11. Impaired mouse mammary gland growth and development is mediated by melatonin and its MT1G protein-coupled receptor via repression of ERα, Akt1, and Stat5.

    PubMed

    Xiang, Shulin; Mao, Lulu; Yuan, Lin; Duplessis, Tamika; Jones, Frank; Hoyle, Gary W; Frasch, Tripp; Dauchy, Robert; Blask, David E; Chakravarty, Geetika; Hill, Steven M

    2012-10-01

    To determine whether melatonin, via its MT(1)  G protein-coupled receptor, impacts mouse mammary gland development, we generated a mouse mammary tumor virus (MMTV)-MT1-Flag-mammary gland over-expressing (MT1-mOE) transgenic mouse. Increased expression of the MT(1) -Flag transgene was observed in the mammary glands of pubescent MT1-mOE transgenic female mice, with further significant increases during pregnancy and lactation. Mammary gland whole mounts from MT1-mOE mice showed significant reductions in ductal growth, ductal branching, and terminal end bud formation. Elevated MT(1) receptor expression in pregnant and lactating female MT1-mOE mice was associated with reduced lobulo-alveolar development, inhibition of mammary epithelial cell proliferation, and significant reductions in body weights of suckling pups. Elevated MT(1) expression in pregnant and lactating MT1-mOE mice correlated with reduced mammary gland expression of Akt1, phospho-Stat5, Wnt4, estrogen receptor alpha, progesterone receptors A and B, and milk proteins β-casein and whey acidic protein. Estrogen- and progesterone-stimulated mammary gland development was repressed by elevated MT(1) receptor expression and exogenous melatonin administration. These studies demonstrate that the MT(1) melatonin receptor and its ligand melatonin play an important regulatory role in mammary gland development and lactation in mice through both growth suppression and alteration of developmental paradigms. © 2012 John Wiley & Sons A/S.

  12. 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

  13. EGR1 expression: a calcium and ERK1/2 mediated PPARγ-independent event involved in the antiproliferative effect of 15-deoxy-Δ12,14-prostaglandin J2 and thiazolidinediones in breast cancer cells.

    PubMed

    Chbicheb, Sarra; Yao, Xiao; Rodeau, Jean-Luc; Salamone, Stéphane; Boisbrun, Michel; Thiel, Gerald; Spohn, Daniel; Grillier-Vuissoz, Isabelle; Chapleur, Yves; Flament, Stéphane; Mazerbourg, Sabine

    2011-05-01

    Our aim was to get new information about the Peroxisome Proliferator Activated Receptor gamma (PPARγ)-independent pathway involved in the antiproliferative action of PPARγ ligands in breast cancer cells. We investigated the effects of Troglitazone (TGZ), Ciglitazone (CGZ), Rosiglitazone (RGZ) and, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ(2)) on the hormone-dependent breast cancer cell line MCF7. The early transcription factor EGR1 (Early Growth Response gene 1) mRNA and protein levels peaked after 3h of incubation with 25μM TGZ, CGZ or 15d-PGJ(2) and then gradually decreased. RGZ, the most potent activator of PPARγ, did not show this effect. The PPARγ antagonist GW 9662 did not block EGR1 mRNA induction which also still occurred in case of PPARγ silencing as well as in case of treatment with the PPARγ-inactive compound Δ2-TGZ. EGR1 mRNA induction required ERK1/2 phosphorylation which was not blocked by EGF Receptor (EGFR) inhibition. The ERK1/2 pathway was also involved in Δ2-TGZ-induced EGR1 mRNA expression in the hormone-independent breast cancer cell line MDA-MB-231. Using the fluorescent dye Fura2, we showed in MCF7 that TGZ or Δ2-TGZ induced an immediate increase in cytosolic calcium which was required for ERK1/2 phosphorylation and EGR1 mRNA induction as demonstrated by calcium chelation experiments. Furthermore, in MCF7 transfected with siRNA targeting EGR1, Δ2-TGZ inhibited less efficiently cell proliferation. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. 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

  15. Identification of the minimal repression domain of SUPERMAN shows that the DLELRL hexapeptide is both necessary and sufficient for repression of transcription in Arabidopsis.

    PubMed

    Hiratsu, Keiichiro; Mitsuda, Nobutaka; Matsui, Kyoko; Ohme-Takagi, Masaru

    2004-08-13

    We reported previously that the carboxy-terminal 30 amino acids of SUPERMAN (SUPRD) function as a repression domain in Arabidopsis. In this study, we identified the peptide sequences in SUPRD that is both necessary and sufficient for repression of transcription. To our surprise, the hexapeptide DLELRL was sufficient, by itself, to confer the ability to repress transcription on a DNA-binding domain. A database search revealed that there are 32 TFIIIA-type zinc finger proteins in the Arabidopsis genome that contain a hexapeptide sequence similar or identical to that of DLELRL. These peptides acted as repression domains, suggesting that these zinc finger proteins might function as active repressors. Further mutational analysis within DLELRL revealed that an amphiphilic motif composed of six amino acids (XLxLXL) with preferences at the first and fifth positions is necessary and sufficient for strong repression. An assay of positional effects suggested that GAL4DB-DLELRL might function as a short-range repressor. A possible mechanism of the DLELRL-mediated repression is discussed.

  16. Something Silent This Way Forms: The Functional Organization of the Repressive Nuclear Compartment

    PubMed Central

    Ritland Politz, Joan C.; Scalzo, David; Groudine, Mark

    2014-01-01

    The repressive compartment of the nucleus is comprised primarily of telomeric and centromeric regions, the silent portion of ribosomal RNA genes, the majority of transposable element repeats, and facultatively repressed genes specific to different cell types. This compartment localizes into three main regions: the peripheral heterochromatin, perinucleolar heterochromatin, and pericentromeric heterochromatin. Both chromatin remodeling proteins and transcription of noncoding RNAs are involved in maintenance of repression in these compartments. Global reorganization of the repressive compartment occurs at each cell division, during early development, and during terminal differentiation. Differential action of chromatin remodeling complexes and boundary element looping activities are involved in mediating these organizational changes. We discuss the evidence that heterochromatin formation and compartmentalization may drive nuclear organization. PMID:23834025

  17. Reciprocal repression between P53 and TCTP.

    PubMed

    Amson, Robert; Pece, Salvatore; Lespagnol, Alexandra; Vyas, Rajesh; Mazzarol, Giovanni; Tosoni, Daniela; Colaluca, Ivan; Viale, Giuseppe; Rodrigues-Ferreira, Sylvie; Wynendaele, Jessika; Chaloin, Olivier; Hoebeke, Johan; Marine, Jean-Christophe; Di Fiore, Pier Paolo; Telerman, Adam

    2011-12-11

    Screening for genes that reprogram cancer cells for the tumor reversion switch identified TCTP (encoding translationally controlled tumor protein) as a crucial regulator of apoptosis. Here we report a negative feedback loop between P53 and TCTP. TCTP promotes P53 degradation by competing with NUMB for binding to P53-MDM2-containing complexes. TCTP inhibits MDM2 auto-ubiquitination and promotes MDM2-mediated ubiquitination and degradation of P53. Notably, Tctp haploinsufficient mice are sensitized to P53-dependent apoptosis. In addition, P53 directly represses TCTP transcription. In 508 breast cancers, high-TCTP status associates with poorly differentiated, aggressive G3-grade tumors, predicting poor prognosis (P < 0.0005). Tctp knockdown in primary mammary tumor cells from ErbB2 transgenic mice results in increased P53 expression and a decreased number of stem-like cancer cells. The pharmacological compounds sertraline and thioridazine increase the amount of P53 by neutralizing TCTP's action on the MDM2-P53 axis. This study links TCTP and P53 in a previously unidentified regulatory circuitry that may underlie the relevance of TCTP in cancer.

  18. 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

  19. Repression of the Histidine Operon: Effect of the First Enzyme on the Kinetics of Repression

    PubMed Central

    Kovach, John S.; Berberich, M. A.; Venetianer, Pál; Goldberger, Robert F.

    1969-01-01

    Kinetic studies on repression of the enzymes for histidine biosynthesis in Salmonella typhimurium showed that, upon addition of histidine to a derepressed culture, the enzymes became repressed in a temporal sequence which corresponds with the positional sequence of the genes in the histidine operon. This serial pattern of repression occurred under conditions in which the feedback site of the first enzyme for histidine biosynthesis is intact. When this site was rendered nonfunctional the pattern of repression was changed so that all of the enzymes became repressed concomitantly. These results suggest that the first enzyme for histidine biosynthesis plays a hitherto unrecognized role in control of the histidine system. PMID:4887508

  20. 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.

  1. Molecular basis for the direct inhibition of AP-1 DNA binding by 15-deoxy-Delta 12,14-prostaglandin J2.

    PubMed

    Pérez-Sala, Dolores; Cernuda-Morollón, Eva; Cañada, F Javier

    2003-12-19

    Cyclopentenone prostaglandins may interfere with cellular functions by multiple mechanisms. The cyclopentenone 15-deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2) has been reported to inhibit the activity of the transcription factor AP-1 in several experimental settings. We have explored the possibility of a direct interaction of 15d-PGJ2 with AP-1 proteins. Here we show that 15d-PGJ2 covalently modifies c-Jun and directly inhibits the DNA binding activity of AP-1. The modification of c-Jun occurs both in vitro and in intact cells as detected by labeling with biotinylated 15d-PGJ2 and mass spectrometry analysis. Attachment of the cyclopentenone prostaglandin occurs at cysteine 269, which is located in the c-Jun DNA binding domain. In addition, 15d-PGJ2 can promote the oligomerization of a fraction of c-Jun through the formation of intermolecular disulfide bonds or 15d-PGJ2-bonded dimers. Our results identify a novel site of interaction of 15d-PGJ2 with the AP-1 activation pathway that may contribute to the complex effects of cyclopentenone prostaglandins on the cellular response to pro-inflammatory agents. They also show the first evidence for the induction of protein cross-linking by 15d-PGJ2.

  2. Contribution of covalent protein modification to the antiinflammatory effects of cyclopentenone prostaglandins.

    PubMed

    Pérez-Sala, Dolores; Cernuda-Morollón, Eva; Pineda-Molina, Estela; Cañada, F Javier

    2002-11-01

    Cyclopentenone prostaglandins, which are produced during inflammatory processes, may exert a negative feedback on inflammation. These reactive compounds may form covalent adducts with thiol groups in glutathione or in proteins. The transcription factor NF-kappaB is key for the expression of numerous proinflammatory genes. We have observed that treatment of mesangial cells with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) inhibits the cytokine-elicited DNA binding activity of NF-kappaB, both in intact cells and in isolated nuclear extracts, thus suggesting a direct effect on DNA binding. By using a biotinylated 15d-PGJ(2) derivative, we have observed that 15d-PGJ(2) forms an adduct with the p50 subunit of NF-kappaB, as shown by Western blot and detection with horseradish peroxidase-conjugated streptavidin. In contrast, a p50 construct that bears a mutation in the cysteine residue involved in DNA binding (Cys62Ser) and is not susceptible to inhibition by 15d-PGJ(2) does not incorporate biotinylated 15d-PGJ(2). The labeling of several polypeptides after incubation of cells with biotinylated 15d-PGJ(2) suggests that there may be multiple targets for modification by 15d-PGJ(2). We propose that the covalent modification of NF-kappaB (and potentially other proteins) by 15d-PGJ(2) may contribute to the antiinflammatory effects of this prostaglandin.

  3. Neuron type-specific miRNA represses two broadly expressed genes to modulate an avoidance behavior in C. elegans

    PubMed Central

    Drexel, Tanja; Mahofsky, Katharina; Latham, Richard; Zimmer, Manuel

    2016-01-01

    Two broad gene classes are distinguished within multicellular organisms: cell type-specific genes, which confer particular cellular properties, and ubiquitous genes that support general cellular functions. However, certain so-called ubiquitous genes show functionally relevant cell type-specific repression. How such repression is achieved is poorly understood. MicroRNAs (miRNAs) are repressors, many of which are expressed with high cell type specificity. Here we show that mir-791, expressed exclusively in the CO2-sensing neurons in Caenorhabditis elegans, represses two otherwise broadly expressed genes. This repression is necessary for normal neuronal function and behavior of the animals toward CO2. miRNA-mediated repression of broadly transcribed genes is a previously unappreciated strategy for cellular specialization. PMID:27688400

  4. 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

  5. mRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons

    PubMed Central

    Jakobson, M; Lintulahti, A; Arumäe, U

    2012-01-01

    mRNA for neuronal Bak (N-Bak), a splice variant of pro-apoptotic Bcl-2 family member Bak is expressed in the neurons. Surprisingly the endogeneous N-Bak protein cannot be demonstrated in the neurons, although the antibodies recognize N-Bak protein from in vitro translation or transiently transfected cells. As N-Bak mRNA contains premature termination codon (PTC) at 89 nucleotides upstream from the last exon–exon junction, it could be degraded by nonsense-mediated decay (NMD) during the pioneer round of translation thus explaining the absence of the protein. We show here that the endogeneous neuronal N-Bak mRNA is not the NMD substrate, as it is not accumulating by cycloheximide treatment, it has a long lifetime, and even prevention of PTC by interfering with the alternative splicing did not lead to translation of the Bak mRNA. N-Bak protein is also not revealed by proteasome inhibitors. Our data suggest strong translational arrest of N-Bak mRNA in the neurons. We show that this arrest is partially mediated by 5′-untranslated region of Bak mRNA and it is not released during mitochondrial apoptosis. PMID:22297299

  6. mRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons.

    PubMed

    Jakobson, M; Lintulahti, A; Arumäe, U

    2012-02-02

    mRNA for neuronal Bak (N-Bak), a splice variant of pro-apoptotic Bcl-2 family member Bak is expressed in the neurons. Surprisingly the endogeneous N-Bak protein cannot be demonstrated in the neurons, although the antibodies recognize N-Bak protein from in vitro translation or transiently transfected cells. As N-Bak mRNA contains premature termination codon (PTC) at 89 nucleotides upstream from the last exon-exon junction, it could be degraded by nonsense-mediated decay (NMD) during the pioneer round of translation thus explaining the absence of the protein. We show here that the endogeneous neuronal N-Bak mRNA is not the NMD substrate, as it is not accumulating by cycloheximide treatment, it has a long lifetime, and even prevention of PTC by interfering with the alternative splicing did not lead to translation of the Bak mRNA. N-Bak protein is also not revealed by proteasome inhibitors. Our data suggest strong translational arrest of N-Bak mRNA in the neurons. We show that this arrest is partially mediated by 5'-untranslated region of Bak mRNA and it is not released during mitochondrial apoptosis.

  7. Mitosis-associated repression in development

    PubMed Central

    Esposito, Emilia; Lim, Bomyi; Guessous, Ghita; Falahati, Hanieh; Levine, Michael

    2016-01-01

    Transcriptional repression is a pervasive feature of animal development. Here, we employ live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. We propose that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail. PMID:27401553

  8. Expression of the Xylulose 5-Phosphate Phosphoketolase Gene, xpkA, from Lactobacillus pentosus MD363 Is Induced by Sugars That Are Fermented via the Phosphoketolase Pathway and Is Repressed by Glucose Mediated by CcpA and the Mannose Phosphoenolpyruvate Phosphotransferase System

    PubMed Central

    Posthuma, Clara C.; Bader, Rechien; Engelmann, Roswitha; Postma, Pieter W.; Hengstenberg, Wolfgang; Pouwels, Peter H.

    2002-01-01

    Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described. xpkA encodes a 788-amino-acid protein with a calculated mass of 88,705 Da. Expression of xpkA in Escherichia coli led to an increase in XpkA activity, while an xpkA knockout mutant of L. pentosus lost XpkA activity and was not able to grow on energy sources that are fermented via the PKP, indicating that xpkA encodes an enzyme with phosphoketolase activity. A database search revealed that there are high levels of similarity between XpkA and a phosphoketolase from Bifidobacterium lactis and between XpkA and a (putative) protein present in a number of evolutionarily distantly related organisms (up to 54% identical residues). Expression of xpkA in L. pentosus was induced by sugars that are fermented via the PKP and was repressed by glucose mediated by carbon catabolite protein A (CcpA) and by the mannose phosphoenolpyruvate phosphotransferase system. Most of the residues involved in correct binding of the cofactor thiamine pyrophosphate (TPP) that are conserved in transketolase, pyruvate decarboxylase, and pyruvate oxidase were also conserved at a similar position in XpkA, implying that there is a similar TPP-binding fold in XpkA. PMID:11823225

  9. 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

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

    PubMed

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

    2015-10-01

    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.

  11. 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

  12. Plant stem cell maintenance involves direct transcriptional repression of differentiation program.

    PubMed

    Yadav, Ram Kishor; Perales, Mariano; Gruel, Jérémy; Ohno, Carolyn; Heisler, Marcus; Girke, Thomas; Jönsson, Henrik; Reddy, G Venugopala

    2013-01-01

    In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.

  13. 15-Deoxy-Delta 12,14-prostaglandin J2 inhibition of NF-kappaB-DNA binding through covalent modification of the p50 subunit.

    PubMed

    Cernuda-Morollón, E; Pineda-Molina, E; Cañada, F J; Pérez-Sala, D

    2001-09-21

    Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF-kappaB. Here we explore the possibility that the NF-kappaB subunit p50 may be a target for the cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)). This prostaglandin inhibited the DNA binding ability of recombinant p50 in a dose-dependent manner. The inhibition required the cyclopentenone moiety and could be prevented but not reverted by glutathione and dithiothreitol. Moreover, a p50 mutant with a C62S mutation was resistant to inhibition, indicating that the effect of 15d-PGJ(2) was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by 15d-PGJ(2) was demonstrated by reverse-phase high pressure liquid chromatography and mass spectrometry analysis that showed an increase in retention time and in the molecular mass of 15d-PGJ(2)-treated p50, respectively. The interaction between p50 and 15d-PGJ(2) was relevant in intact cells. 15d-PGJ(2) effectively inhibited cytokine-elicited NF-kappaB activity in HeLa without reducing IkappaBalpha degradation or nuclear translocation of NF-kappaB subunits. 15d-PGJ(2) reduced NF-kappaB DNA binding activity in isolated nuclear extracts, suggesting a direct effect on NF-kappaB proteins. Finally, treatment of HeLa with biotinylated-15d-PGJ(2) resulted in the formation of a 15d-PGJ(2)-p50 adduct as demonstrated by neutravidin binding and immunoprecipitation. These results clearly show that p50 is a target for covalent modification by 15d-PGJ(2) that results in inhibition of DNA binding.

  14. Pseudomonas fluorescens WCS374r-induced systemic resistance in rice against Magnaporthe oryzae is based on pseudobactin-mediated priming for a salicylic acid-repressible multifaceted defense response.

    PubMed

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

    2008-12-01

    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

  15. Fisetin Suppresses Lipid Accumulation in Mouse Adipocytic 3T3-L1 Cells by Repressing GLUT4-Mediated Glucose Uptake through Inhibition of mTOR-C/EBPα Signaling.

    PubMed

    Watanabe, Marina; Hisatake, Mitsuhiro; Fujimori, Ko

    2015-05-27

    3,7,3',4'-Tetrahydroxyflavone (fisetin) is a flavonoid found in vegetables and fruits having broad biological activities. Here the effects of fisetin on adipogenesis and its regulatory mechanism in mouse adipocytic 3T3-L1 cells are studied. Fisetin inhibited the accumulation of intracellular lipids and lowered the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein (C/EBP) α and fatty acid-binding protein 4 (aP2) during adipogenesis. Moreover, the mRNA levels of genes such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase involved in the fatty acid biosynthesis (lipogenesis) were reduced by the treatment with fisetin. The expression level of the glucose transporter 4 (GLUT4) gene was also decreased by fisetin, resulting in down-regulation of glucose uptake. Furthermore, fisetin inhibited the phosphorylation of the mammalian target of rapamycin (mTOR) and that of p70 ribosomal S6 kinase, a target of the mTOR complex, the inhibition of which was followed by a decreased mRNA level of the C/EBPα gene. The results obtained from a chromatin immunoprecipitation assay demonstrated that the ability of C/EBPα to bind to the GLUT4 gene promoter was reduced by the treatment with fisetin, which agreed well with those obtained when 3T3-L1 cells were allowed to differentiate into adipocytes in medium in the presence of rapamycin, an inhibitor for mTOR. These results indicate that fisetin suppressed the accumulation of intracellular lipids by inhibiting GLUT4-mediated glucose uptake through inhibition of the mTOR-C/EBPα signaling in 3T3-L1 cells.

  16. Two Distinct Repressive Mechanisms for Histone 3 Lysine 4 Methylation through Promoting 3′-End Antisense Transcription

    PubMed Central

    Margaritis, Thanasis; Oreal, Vincent; Brabers, Nathalie; Maestroni, Laetitia; Vitaliano-Prunier, Adeline; Benschop, Joris J.; van Hooff, Sander; van Leenen, Dik

    2012-01-01

    Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3′-end, indicating that repression is coupled to 3′-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3′-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3′-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3′-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3′-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4

  17. Hepatocyte nuclear factor-3 homologue 1 (HFH-1) represses transcription of smooth muscle-specific genes.

    PubMed

    Hoggatt, A M; Kriegel, A M; Smith, A F; Herring, B P

    2000-10-06

    Results show that smooth muscle-specific promoters represent novel downstream targets of the winged helix factor hepatocyte nuclear factor-3 homologue 1 (HFH-1). HFH-1 strongly represses telokin promoter activity when overexpressed in A10 vascular smooth muscle cells. HFH-1 was also found to repress transcription of several other smooth muscle-specific promoters, including the SM22alpha promoter. HFH-1 inhibits telokin promoter activity, by binding to a forkhead consensus site located within an AT-rich region of the telokin promoter. The DNA-binding domain alone was sufficient to mediate inhibition, suggesting that binding of HFH-1 blocks the binding of other positive-acting factors. HFH-1 does not disrupt serum response factor binding to an adjacent CArG box within the telokin promoter, implying that HFH-1 must compete with other unidentified trans-activators to mediate repression. The localization of HFH-1 mRNA to the epithelial cell layer of mouse bladder and stomach implicates HFH-1 in repressing telokin expression in epithelial cells. This suggests that cell-specific expression of telokin is likely mediated by both positive-acting factors in smooth muscle cells and negative-acting factors in nonmuscle cell types. We propose a model in which the smooth muscle specificity of the telokin promoter is regulated by interactions between positive- and negative-acting members of the hepatocyte nuclear factor-3/forkhead family of transcription factors.

  18. 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

    2015-07-23

    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.

  19. Transcriptional repression of the tumor suppressor DRO1 by AIB1.

    PubMed

    Ferragud, Juan; Avivar-Valderas, Alvaro; Pla, Antoni; De Las Rivas, Javier; Font de Mora, Jaime

    2011-10-03

    Using transcriptomic gene expression profiling we found tumor suppressor DRO1 being repressed in AIB1 transgenic mice. In agreement, AIB1 represses DRO1 promoter and its expression levels inversely correlate with DRO1 in several cancer cell lines and in ectopic and silencing assays. Estrogen modulators treatment showed a regulation in an estrogen receptor-dependent fashion. Importantly, DRO1 overexpression resulted in BCLAF1 upregulation, a compelling concept given that BCLAF1 is a death-promoting transcriptional repressor. Additionally, DRO1 shuttles from Golgi to the endoplasmic reticulum upon apoptotic stimuli, where it is predicted to facilitate the apoptosis cascade. Finally, DRO1 repression is an important factor for AIB1-mediated inhibition of apoptosis. Collectively, our results reveal DRO1 as an AIB1-targeted tumor suppressor, providing a novel mechanism for AIB1-dependent inhibition of apoptosis. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  20. Hormonal regulation of TSE1-repressed genes: evidence for multiple genetic controls in extinction.

    PubMed Central

    Thayer, M J; Fournier, R E

    1989-01-01

    Somatic cell hybrids formed by fusing hepatoma cells with fibroblasts generally fail to express liver functions, a phenomenon termed extinction. Previous studies demonstrated that extinction of the genes encoding tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and argininosuccinate synthetase is mediated by a specific genetic locus (TSE1) that maps to mouse chromosome 11 and human chromosome 17. In this report, we show that full repression of these genes requires a genetic factor in addition to TSE1. This conclusion is based on the observation that residual gene activity was apparent in monochromosomal hybrids retaining human TSE1 but not in complex hybrids retaining many fibroblast chromosomes. Furthermore, TSE1-repressed genes were hormone inducible, whereas fully extinguished genes were not. Analysis of hybrid segregants indicated that genetic loci required for the complete repression phenotype were distinct from TSE1. Images PMID:2571076

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

    PubMed Central

    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

  2. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD.

    PubMed

    Ling, Jonathan P; Pletnikova, Olga; Troncoso, Juan C; Wong, Philip C

    2015-08-07

    Cytoplasmic aggregation of TDP-43, accompanied by its nuclear clearance, is a key common pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, a limited understanding of this RNA-binding protein (RBP) impedes the clarification of pathogenic mechanisms underlying TDP-43 proteinopathy. In contrast to RBPs that regulate splicing of conserved exons, we found that TDP-43 repressed the splicing of nonconserved cryptic exons, maintaining intron integrity. When TDP-43 was depleted from mouse embryonic stem cells, these cryptic exons were spliced into messenger RNAs, often disrupting their translation and promoting nonsense-mediated decay. Moreover, enforced repression of cryptic exons prevented cell death in TDP-43-deficient cells. Furthermore, repression of cryptic exons was impaired in ALS-FTD cases, suggesting that this splicing defect could potentially underlie TDP-43 proteinopathy.

  3. Mitosis-associated repression in development.

    PubMed

    Esposito, Emilia; Lim, Bomyi; Guessous, Ghita; Falahati, Hanieh; Levine, Michael

    2016-07-01

    Transcriptional repression is a pervasive feature of animal development. Here, we employ live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. We propose that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail. © 2016 Esposito et al.; Published by Cold Spring Harbor Laboratory Press.

  4. Molecular architecture of polycomb repressive complexes

    PubMed Central

    Chittock, Emily C.; Latwiel, Sebastian; Miller, Thomas C.R.

    2017-01-01

    The polycomb group (PcG) proteins are a large and diverse family that epigenetically repress the transcription of key developmental genes. They form three broad groups of polycomb repressive complexes (PRCs) known as PRC1, PRC2 and Polycomb Repressive DeUBiquitinase, each of which modifies and/or remodels chromatin by distinct mechanisms that are tuned by having variable compositions of core and accessory subunits. Until recently, relatively little was known about how the various PcG proteins assemble to form the PRCs; however, studies by several groups have now allowed us to start piecing together the PcG puzzle. Here, we discuss some highlights of recent PcG structures and the insights they have given us into how these complexes regulate transcription through chromatin. PMID:28202673

  5. PHB biosynthesis in catabolite repression mutant of Burkholderia sacchari.

    PubMed

    Lopes, Mateus Schreiner Garcez; Gosset, Guillermo; Rocha, Rafael Costa Santos; Gomez, José Gregório Cabrera; Ferreira da Silva, Luiziana

    2011-10-01

    Due to the effect of catabolite repression, sugar mixtures cannot be metabolized in a rapid and efficient way implicating in lower productivity in bioprocesses using lignocellulosic hydrolysates. In gram-negative bacteria, this mechanism is mediated by the phosphotransferase system (PTS), which concomitantly internalizes and phosphorylates sugars. In this study, we isolated a UV mutant of Burkholderia sacchari, called LFM828, which transports hexoses and pentoses by a non-PTS uptake system. This mutant presented released glucose catabolite repression over the pentoses. In mixtures of glucose, xylose, and arabinose, specific growth rates and the specific sugar consumption rates were, respectively, 10 and 23% higher in LFM828, resulting in a reduced time to exhaust all sugars in the medium. However, in polyhydroxybutyrate (PHB) biosynthesis experiments it was necessary the supplementation of yeast extract to maintain higher values of growth rate and sugar consumption rate. The deficient growth in mineral medium was partially recovered by replacing the ammonium nitrogen source by glutamate. It was demonstrated that the ammonium metabolism is not defective in LFM828, differently from ammonium, glutamate can also be used as carbon and energy allowing an improvement on the carbohydrates utilization for PHB production in LFM828. In contrast, higher rates of ammonia consumption and CO(2) production in LFM828 indicate altered fluxes through the central metabolism in LFM828 and the parental. In conclusion, PTS plays an important role in cell physiology and the elimination of its components has a significant impact on catabolite repression, carbon flux distribution, and PHB biosynthesis in B. sacchari.

  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. Protein thiol modification by 15-deoxy-Delta12,14-prostaglandin J2 addition in mesangial cells: role in the inhibition of pro-inflammatory genes.

    PubMed

    Sánchez-Gómez, Francisco J; Cernuda-Morollón, Eva; Stamatakis, Konstantinos; Pérez-Sala, Dolores

    2004-11-01

    The cyclopentenone prostaglandin and PPARgamma agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) displays anti-inflammatory effects in several experimental models. Direct modification of protein thiols is arising as an important mechanism of cyclopentenone prostaglandin action. However, little is known about the extent or specificity of this process. Mesangial cells (MC) play a key role in glomerulonephritis. In this work, we have studied the selectivity of protein modification by 15d-PGJ(2) in MC, and the correlation with the modulation of several proinflammatory genes. MC incubation with biotinylated 15d-PGJ(2) results in the labeling of a distinct set of proteins as evidenced by two-dimensional electrophoresis. 15d-PGJ(2) binds to nuclear and cytosolic targets as detected by fluorescence microscopy and subcellular fractionation. The pattern of biotinylated 15d-PGJ(2)-modified polypeptides is readily distinguishable from that of total protein staining or labeling with biotinylated iodoacetamide. 15d-PGJ(2) addition requires the double bond in the cyclopentane ring. 9,10-Dihydro-15d-PGJ(2), a 15d-PGJ(2) analog that shows the same potency as peroxisome proliferator-activated receptor (PPAR) agonist in MC but lacks the cyclopentenone moiety, displays reduced ability to modify proteins and to block 15d-PGJ(2) binding. Micromolar concentrations of 15d-PGJ(2) inhibit cytokine-elicited levels of inducible nitricoxide synthase, cyclooxygenase-2, and intercellular adhesion molecule-1 in MC. In contrast, 9,10-dihydro-15d-PGJ(2) does not reproduce this inhibition. 15d-PGJ(2) effect is not blocked by the PPARgamma antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Moreover, compounds possessing an alpha,beta-unsaturated carbonyl group, like 2-cyclopenten-1-one and 2-cyclohexen-1-one, reduce pro-inflammatory gene expression. These observations indicate that covalent modification of cellular thiols by 15d-PGJ(2) is a selective process that plays an important

  8. Electrophilic peroxisome proliferator-activated receptor-gamma ligands have potent antifibrotic effects in human lung fibroblasts.

    PubMed

    Ferguson, Heather E; Kulkarni, Ajit; Lehmann, Geniece M; Garcia-Bates, Tatiana M; Thatcher, Thomas H; Huxlin, Krystel R; Phipps, Richard P; Sime, Patricia J

    2009-12-01

    Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-beta is up-regulated in fibrotic diseases, where it stimulates differentiation of fibroblasts to myofibroblasts and production of excess extracellular matrix. Peroxisome proliferator-activated receptor (PPAR) gamma is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPARgamma ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-beta-stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of alpha-smooth muscle actin, fibronectin, collagen, and the novel myofibroblast marker, calponin. The inhibitory concentration causing a 50% decrease in aSMA for CDDO was 20-fold lower than the endogenous PPARgamma ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15 d-PGJ(2)), and 400-fold lower than the synthetic ligand, rosiglitazone. Pharmacologic and genetic approaches were used to demonstrate that CDDO mediates its activity via a PPARgamma-independent pathway. CDDO and 15 d-PGJ(2) contain an alpha/beta unsaturated ketone, which acts as an electrophilic center that can form covalent bonds with cellular proteins. Prostaglandin A(1) and diphenyl diselenide, both strong electrophiles, also inhibit myofibroblast differentiation, but a structural analog of 15 d-PGJ(2) lacking the electrophilic center is much less potent. CDDO does not alter TGF-beta-induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-beta-responsive genes. Overall, these data indicate that certain PPARgamma ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-beta-mediated profibrogenic activities through pathways independent of PPARgamma. As the inhibitory concentration causing a 50% decrease in a

  9. Obacunone Represses Salmonella Pathogenicity Islands 1 and 2 in an envZ-Dependent Fashion

    PubMed Central

    Vikram, Amit; Jayaprakasha, Guddadarangavvanahally K.; Jesudhasan, Palmy R.

    2012-01-01

    Obacunone belongs to a class of unique triterpenoids called limonoids, present in Citrus species. Previous studies from our laboratory suggested that obacunone possesses antivirulence activity and demonstrates inhibition of cell-cell signaling in Vibrio harveyi and Escherichia coli O157:H7. The present work sought to determine the effect of obacunone on the food-borne pathogen Salmonella enterica serovar Typhimurium LT2 by using a cDNA microarray. Transcriptomic studies indicated that obacunone represses Salmonella pathogenicity island 1 (SPI1), the maltose transporter, and the hydrogenase operon. Furthermore, phenotypic data for the Caco-2 infection assay and maltose utilization were in agreement with microarray data suggesting repression of SPI1 and maltose transport. Further studies demonstrated that repression of SPI1 was plausibly mediated through hilA. Additionally, obacunone seems to repress SPI2 under SPI2-inducing conditions as well as in Caco-2 infection models. Furthermore, obacunone seems to repress hilA in an EnvZ-dependent fashion. Altogether, the results of the study seems to suggest that obacunone exerts an antivirulence effect on S. Typhimurium and may serve as a lead compound for development of antivirulence strategies for S. Typhimurium. PMID:22843534

  10. Evidence for ORC-dependent repression of budding yeast genes induced by starvation and other stresses.

    PubMed

    Ramachandran, Lakshmi; Burhans, Debra T; Laun, Peter; Wang, Jianxin; Liang, Ping; Weinberger, Martin; Wissing, Silke; Jarolim, Stefanie; Suter, Bernhard; Madeo, Frank; Breitenbach, Michael; Burhans, William C

    2006-08-01

    The highly conserved origin recognition complex (ORC) is required for repressing genes in the silent mating type loci of budding yeast. Here we report that at a non-permissive temperature, the temperature-sensitive orc2-1 mutation induces the expression of more than 500 genes, the majority of which are also induced during starvation of wild-type cells. Many genes induced by starvation or by the orc2-1 mutation are also induced by inactivation of proteins required for chromatin-mediated repression of transcription. Genes induced by the orc2-1 mutation, starvation, or inactivation of repressor proteins, map near ORC-binding loci significantly more frequently compared to all genes. Genes repressed by starvation map near ORC-binding sites less frequently compared to all genes, which suggests they have been evolutionarily excluded from regions of repressive chromatin near ORC-binding sites. Deletion of sequences containing ORC-binding sites near the DAL2 and DAL4 genes in the DAL gene cluster, which are induced by either the orc2-1 mutation or by starvation, constitutively activates these genes and abolishes their activation by the orc2-1 mutation. Our findings suggest a role for ORC in the repression of a large number of budding yeast genes induced by starvation or other aspects of a deleterious environment.

  11. Splicing repression allows the gradual emergence of new Alu-exons in primate evolution

    PubMed Central

    Attig, Jan; Ruiz de los Mozos, Igor; Haberman, Nejc; Wang, Zhen; Emmett, Warren; Zarnack, Kathi; König, Julian; Ule, Jernej

    2016-01-01

    Alu elements are retrotransposons that frequently form new exons during primate evolution. Here, we assess the interplay of splicing repression by hnRNPC and nonsense-mediated mRNA decay (NMD) in the quality control and evolution of new Alu-exons. We identify 3100 new Alu-exons and show that NMD more efficiently recognises transcripts with Alu-exons compared to other exons with premature termination codons. However, some Alu-exons escape NMD, especially when an adjacent intron is retained, highlighting the importance of concerted repression by splicing and NMD. We show that evolutionary progression of 3' splice sites is coupled with longer repressive uridine tracts. Once the 3' splice site at ancient Alu-exons reaches a stable phase, splicing repression by hnRNPC decreases, but the exons generally remain sensitive to NMD. We conclude that repressive motifs are strongest next to cryptic exons and that gradual weakening of these motifs contributes to the evolutionary emergence of new alternative exons. DOI: http://dx.doi.org/10.7554/eLife.19545.001 PMID:27861119

  12. Active Repression of Methylated Genes by the Chromosomal Protein MBD1

    PubMed Central

    Ng, Huck-Hui; Jeppesen, Peter; Bird, Adrian

    2000-01-01

    MBD1 belongs to a family of mammalian proteins that share a methyl-CpG binding domain. Previous work has shown that MBD1 binds to methylated sites in vivo and in vitro and can repress transcription from methylated templates in transcription extracts and in cultured cells. In the present study we established by several experimental criteria that, contrary to a previous report, MBD1 is not a component of the MeCP1 repressor complex. We identified a powerful transcriptional repression domain (TRD) at the C terminus of MBD1 that can actively repress transcription at a distance. Methylation-dependent repression in vivo depends on the presence of both the TRD and the methyl-CpG binding domain. The mechanism is likely to involve deacetylation, since the deacetylase inhibitor trichostatin A can overcome MBD1-mediated repression. Accordingly, we found that endogenous MBD1 is particularly concentrated at sites of centromeric heterochromatin, where acetylated histone H4 is deficient. Unlike MBD2 and MeCP2, MBD1 is not depleted by antibodies to the histone deacetylase HDAC1. Thus, the deacetylase-dependent pathway by which MBD1 actively silences methylated genes is likely to be different from that utilized by the methylation-dependent repressors MeCP1 and MeCP2. PMID:10648624

  13. zif-1 translational repression defines a second, mutually exclusive OMA function in germline transcriptional repression.

    PubMed

    Guven-Ozkan, Tugba; Robertson, Scott M; Nishi, Yuichi; Lin, Rueyling

    2010-10-01

    Specification of primordial germ cells requires global repression of transcription. In C. elegans, primordial germ cells are generated through four rounds of asymmetric divisions, starting from the zygote P0, each producing a transcriptionally repressed germline blastomere (P1-P4). Repression in P2-P4 requires PIE-1, which is provided maternally in oocytes and segregated to all germline blastomeres. We have shown previously that OMA-1 and OMA-2 repress global transcription in P0 and P1 by sequestering TAF-4, an essential component of TFIID. Soon after the first mitotic cycle, OMA proteins undergo developmentally regulated degradation. Here, we show that OMA proteins also repress transcription in P2-P4 indirectly, through a completely different mechanism that operates in oocytes. OMA proteins bind to both the 3' UTR of the zif-1 transcript and the eIF4E-binding protein, SPN-2, repressing translation of zif-1 mRNA in oocytes. zif-1 encodes the substrate-binding subunit of the E3 ligase for PIE-1 degradation. Inhibition of zif-1 translation in oocytes ensures high PIE-1 levels in oocytes and germline blastomeres. The two OMA protein functions are strictly regulated in both space and time by MBK-2, a kinase activated following fertilization. Phosphorylation by MBK-2 facilitates the binding of OMA proteins to TAF-4 and simultaneously inactivates their function in repressing zif-1 translation. Phosphorylation of OMA proteins displaces SPN-2 from the zif-1 3' UTR, releasing translational repression. We propose that MBK-2 phosphorylation serves as a developmental switch, converting OMA proteins from specific translational repressors in oocytes to global transcriptional repressors in embryos, together effectively repressing transcription in all germline blastomeres.

  14. GWT1 encoding an inositol acyltransferase homolog is required for laccase repression and stress resistance in the basidiomycete Cryptococcus neoformans.

    PubMed

    Zhao, Qiang; Wei, Dongsheng; Li, Zhongming; Wang, Yu; Zhu, Xiangyang; Zhu, Xudong

    2015-12-01

    The transcriptional expression of laccase, which has been confirmed to contribute to the virulence of Cryptococcus neoformans, is often repressed by a high concentration of glucose in many fungi, including C. neoformans. The underlying mechanism of the repression remains largely unknown. In this study, we found that a GWT1 gene that encodes a glycosylphosphatidylinositol (GPI) anchor biosynthesis-related protein is required for laccase repression by glucose in the basidiomycete C. neoformans. Disruption of GWT1 with the Agrobacterium tumefaciens-mediated T-DNA random insertional mutagenesis (ATMT) method resulted in constitutive expression of the laccase gene LAC1 and constant melanin formation. The loss of GWT1 also dramatically affected the cell membrane integrity and stress resistance. Our results revealed a GPI-dependent glucose repression mechanism in C. neoformans, and it may be helpful for understanding the virulence of C. neoformans.

  15. Keratins mediate localization of hemidesmosomes and repress cell motility.

    PubMed

    Seltmann, Kristin; Roth, Wera; Kröger, Cornelia; Loschke, Fanny; Lederer, Marcell; Hüttelmaier, Stefan; Magin, Thomas M

    2013-01-01

    The keratin (K)-hemidesmosome (HD) interaction is crucial for cell-matrix adhesion and migration in several epithelia, including the epidermis. Mutations in constituent proteins cause severe blistering skin disorders by disrupting the adhesion complex. Despite extensive studies, the role of keratins in HD assembly and maintenance is only partially understood. Here we address this issue in keratinocytes in which all keratins are depleted by genome engineering. Unexpectedly, such keratinocytes maintain many characteristics of their normal counterparts. However, the absence of the entire keratin cytoskeleton leads to loss of plectin from the hemidesmosomal plaque and scattering of the HD transmembrane core along the basement membrane zone. To investigate the functional consequences, we performed migration and adhesion assays. These revealed that, in the absence of keratins, keratinocytes adhere much faster to extracellular matrix substrates and migrate approximately two times faster compared with wild-type cells. Reexpression of the single keratin pair K5 and K14 fully reversed the above phenotype. Our data uncover a role of keratins, which to our knowledge is previously unreported, in the maintenance of HDs upstream of plectin, with implications for epidermal homeostasis and pathogenesis. They support the view that the downregulation of keratins observed during epithelial-mesenchymal transition supports the migratory and invasive behavior of tumor cells.

  16. Keratins mediate localization of hemidesmosomes and repress cell motility

    PubMed Central

    Seltmann, Kristin; Roth, Wera; Loschke, Fanny; Lederer, Marcell; Hüttelmaier, Stefan; Magin, Thomas M.

    2013-01-01

    The keratin-hemidesmosome interaction is crucial for cell-matrix adhesion and migration in several epithelia including the epidermis. Mutations in constituent proteins cause severe blistering skin disorders by disrupting the adhesion complex. Despite extensive studies, the role of keratins in hemidesmosome assembly and maintenance is only partially understood. Here, we address this issue in keratinocytes in which all keratins are depleted by genome engineering. Unexpectedly, such keratinocytes maintain many characteristics of normal counterparts. The absence of the entire keratin cytoskeleton, however, leads to loss of plectin from the hemidesmosomal plaque and scattering of the hemidesmosome transmembrane core along the basement membrane zone. To investigate the functional consequences, we performed migration and adhesion assays. These revealed that in the absence of keratins, keratinocytes adhere much faster to ECM substrates and migrate ~2 times faster compared to wildtype cells. Re-expression of the single keratin pair K5 and K14 fully reversed the above phenotype. Our data uncover a novel role of keratins in the maintenance of hemidesmosomes upstream of plectin with implications for epidermal homeostasis and pathogenesis. They support the view that the downregulation of keratins observed during epithelial-mesenchymal transition supports the migratory and invasive behavior of tumor cells. PMID:22895363

  17. Theoretical and Experimental Dissection of DNA Loop-Mediated Repression

    NASA Astrophysics Data System (ADS)

    Boedicker, James Q.; Garcia, Hernan G.; Phillips, Rob

    2013-01-01

    Transcriptional networks across all domains of life feature a wide range of regulatory architectures. Theoretical models now make clear predictions about how key parameters describing those architectures modulate gene expression, and the ability to construct genetic circuits with tunable parameters enables precise tests of such models. We dissect gene regulation through DNA looping by tuning network parameters such as repressor copy number, DNA binding strengths, and loop length in both thermodynamic models and experiments. Our results help clarify the short-length mechanical properties of DNA.

  18. Repression-Sensitization and Health Behavior.

    ERIC Educational Resources Information Center

    Gayton, William F.; And Others

    1978-01-01

    Examined relationship between repression-sensitization (R-S) and visits to prison infirmary for males during a one-year period. Main effect for R-S dimension was significant for total number of visits, number of medically justified visits, and number of medically unjustified visits. Sensitizers had significantly more visits than repressors.…

  19. The great repression: chromatin and cryptic transcription.

    PubMed

    Hennig, Bianca P; Fischer, Tamás

    2013-01-01

    The eukaryotic chromatin structure is essential in correctly defining transcription units. Impairing this structure can activate cryptic promoters, and lead to the accumulation of aberrant RNA transcripts. Here we discuss critical pathways that are responsible for the repression of cryptic transcription and the maintenance of genome integrity.

  20. Trichostatin A enhances estrogen receptor-alpha repression in MCF-7 breast cancer cells under hypoxia

    SciTech Connect

    Noh, Hyunggyun; Park, Joonwoo; Shim, Myeongguk; Lee, YoungJoo

    2016-02-12

    Estrogen receptor (ER) is a crucial determinant of resistance to endocrine therapy, which may change during the progression of breast cancer. We previously showed that hypoxia induces ESR1 gene repression and ERα protein degradation via proteasome-mediated pathway in breast cancer cells. HDAC plays important roles in the regulation of histone and non-histone protein post-translational modification. HDAC inhibitors can induce epigenetic changes and have therapeutic potential for targeting various cancers. Trichostatin A exerts potent antitumor activities against breast cancer cells in vitro and in vivo. In this report, we show that TSA augments ESR1 gene repression at the transcriptional level and downregulates ERα protein expression under hypoxic conditions through a proteasome-mediated pathway. TSA-induced estrogen response element-driven reporter activity in the absence of estrogen was synergistically enhanced under hypoxia; however, TSA inhibited cell proliferation under both normoxia and hypoxia. Our data show that the hypoxia-induced repression of ESR1 and degradation of ERα are enhanced by concomitant treatment with TSA. These findings expand our understanding of hormone responsiveness in the tumor microenvironment; however, additional in-depth studies are required to elucidate the detailed mechanisms of TSA-induced ERα regulation under hypoxia. - Highlights: • TSA augments ESR1 gene repression at the transcriptional level under hypoxia. • TSA downregulates ERα protein expression under hypoxia. • TSA-induced ERα regulation under hypoxia is essential for understanding the behavior and progression of breast cancer.

  1. Translational repression by RNA-binding protein TIAR.

    PubMed

    Mazan-Mamczarz, Krystyna; Lal, Ashish; Martindale, Jennifer L; Kawai, Tomoko; Gorospe, Myriam

    2006-04-01

    The RNA-binding protein TIAR has been proposed to inhibit protein synthesis transiently by promoting the formation of translationally silent stress granules. Here, we report the selective binding of TIAR to several mRNAs encoding translation factors such as eukaryotic initiation factor 4A (eIF4A) and eIF4E (translation initiation factors), eEF1B (a translation elongation factor), and c-Myc (which transcriptionally controls the expression of numerous translation regulatory proteins). TIAR bound the 3'-untranslated regions of these mRNAs and potently suppressed their translation, particularly in response to low levels of short-wavelength UV (UVC) irradiation. The UVC-imposed global inhibition of the cellular translation machinery was significantly relieved after silencing of TIAR expression. We propose that the TIAR-mediated inhibition of translation factor expression elicits a sustained repression of protein biosynthesis in cells responding to stress.

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

    PubMed Central

    Lyons, Shawn M.; Achorn, Chris; Kedersha, Nancy L.; Anderson, Paul J.; Ivanov, Pavel

    2016-01-01

    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

  3. Identification of essential nucleotides in an upstream repressing sequence of Saccharomyces cerevisiae by selection for increased expression of TRK2.

    PubMed Central

    Vidal, M; Buckley, A M; Yohn, C; Hoeppner, D J; Gaber, R F

    1995-01-01

    The TRK2 gene in Saccharomyces cerevisiae encodes a membrane protein involved in potassium transport and is expressed at extremely low levels. Dominant cis-acting mutations (TRK2D), selected by their ability to confer TRK2-dependent growth on low-potassium medium, identified an upstream repressor element (URS1-TRK2) in the TRK2 promoter. The URS1-TRK2 sequence (5'-AGCCGCACG-3') shares six nucleotides with the ubiquitous URS1 element (5'-AGCCGCCGA-3'), and the protein species binding URS1-CAR1 (URSF) is capable of binding URS1-TRK2 in vitro. Sequence analysis of 17 independent repression-defective TRK2D mutations identified three adjacent nucleotides essential for URS1-mediated repression in vivo. Our results suggest a role for context effects with regard to URS1-related sequences: several mutant alleles of the URS1 element previously reported to have little or no effect when analyzed within the context of a heterologous promoter (CYC1) [Luche, R.M., Sumrada, R. & Cooper, T.G. (1990) Mol. Cell. Biol. 10, 3884-3895] have major effects on repression in the context of their native promoters (TRK2 and CAR1). TRK2D mutations that abolish repression also reveal upstream activating sequence activity either within or adjacent to URS1. Additivity between TRK2D and sin3 delta mutations suggest that SIN3-mediated repression is independent of that mediated by URS1. Images Fig. 1 Fig. 4 PMID:7892273

  4. Repression: finding our way in the maze of concepts.

    PubMed

    Garssen, Bert

    2007-12-01

    Repression is associated in the literature with terms such as non-expression, emotional control, rationality, anti-emotionality, defensiveness and restraint. Whether these terms are synonymous with repression, indicate a variation, or are essentially different from repression is uncertain. To clarify this obscured view on repression, this paper indicates the similarities and differences between these concepts. Repression is the general term that is used to describe the tendency to inhibit the experience and the expression of negative feelings or unpleasant cognitions in order to prevent one's positive self-image from being threatened ('repressive coping style'). The terms self-deception versus other-deception, and socially related versus personally related repression refer to what is considered to be different aspects of repression. Defensiveness is a broader concept that includes both anxious defensiveness and repression; the essential difference is whether negative emotions are reported or not. Concepts that are sometimes associated with repression, but which are conceptually different, are also discussed in this paper: The act of suppression, 'repressed memories,' habitual suppression, concealment, type C coping pattern, type D personality, denial, alexithymia and blunting. Consequences for research: (1) When summarizing findings reported in the literature, it is essential to determine which concepts the findings represent. This is rarely made explicit, and failure to do so may lead to drawing the wrong conclusions (2) It is advisable to use scales based on different aspects of repression (3) Whether empirical findings substantiate the similarities and differences between concepts described in this paper will need to be shown.

  5. 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

  6. Insomnia symptoms and repressive coping in a sample of older Black and White women

    PubMed Central

    Jean-Louis, Girardin; Magai, Carol; Consedine, Nathan S; Pierre-Louis, Jessy; Zizi, Ferdinand; Casimir, Georges J; Belzie, Louis

    2007-01-01

    Background This study examined whether ethnic differences in insomnia symptoms are mediated by differences in repressive coping styles. Methods A total of 1274 women (average age = 59.36 ± 6.53 years) participated in the study; 28% were White and 72% were Black. Older women in Brooklyn, NY were recruited using a stratified, cluster-sampling technique. Trained staff conducted face-to-face interviews lasting 1.5 hours acquiring sociodemographic data, health characteristics, and risk factors. A sleep questionnaire was administered and individual repressive coping styles were assessed. Fisher's exact test and Spearman and Pearson analyses were used to analyze the data. Results The rate of insomnia symptoms was greater among White women [74% vs. 46%; χ2 = 87.67, p < 0.0001]. Black women scored higher on the repressive coping scale than did White women [Black = 37.52 ± 6.99, White = 29.78 ± 7.38, F1,1272 = 304.75, p < 0.0001]. We observed stronger correlations between repressive coping and insomnia symptoms for Black [rs = -0.43, p < 0.0001] than for White women [rs = -0.18, p < 0.0001]. Controlling for variation in repressive coping, the magnitude of the correlation between ethnicity and insomnia symptoms was substantially reduced. Multivariate adjustment for differences in sociodemographics, health risk factors, physical health, and health beliefs and attitudes had little effect on the relationships. Conclusion Relationships between ethnicity and insomnia symptoms are jointly dependent on the degree of repressive coping, suggesting that Black women may be reporting fewer insomnia symptoms because of a greater ability to route negative emotions from consciousness. It may be that Blacks cope with sleep problems within a positive self-regulatory framework, which allows them to deal more effectively with sleep-interfering psychological processes to stressful life events and to curtail dysfunctional sleep-interpreting processes. PMID:17261187

  7. Possible involvement of aiPLA2 in the phosphatidylserine-containing liposomes induced production of PGE2 and PGD2 in microglia.

    PubMed

    Takayama, Fumiko; Wu, Zhou; Ma, Hong Mei; Okada, Ryo; Hayashi, Yoshinori; Nakanishi, Hiroshi

    2013-09-15

    Liposomes containing phosphatidylserine (PSL) produce PGE2 after being phagocytosed by microglia, but the precise underlying mechanism behind it still remains unclear. Here, we showed that liposomes consisting of phosphatidylserine and lysophosphatidylcholine, a lipolysis product of phosphatidylcholine by PLA2, were phagocytosed by microglia, but failed to induce secretion of PGE2. Furthermore, PSL-induced PGE2 secretion was significantly inhibited by MJ33, an aiPLA2 inhibitor, but not by AACOCF3, a cPLA2 inhibitor. PSL also produced PGD2 and 15d-PGJ2 in microglia. We thus hypothesize that free arachidonic acid is supplied through aiPLA2-mediated lipolysis of phagocytosed phosphatidylcholine, leading to the production of PGH2 and its downstream metabolites.

  8. TORC2 regulates germinal center repression of the TCL1 oncoprotein to promote B cell development and inhibit transformation.

    PubMed

    Kuraishy, Ali I; French, Samuel W; Sherman, Mara; Herling, Marco; Jones, Dan; Wall, Randolph; Teitell, Michael A

    2007-06-12

    Aberrant expression of the TCL1 oncoprotein promotes malignant transformation of germinal center (GC) B cells. Repression of TCL1 in GC B cells facilitates FAS-mediated apoptosis and prevents lymphoma formation. However, the mechanism for this repression is unknown. Here we show that the CREB coactivator TORC2 directly regulates TCL1 expression independent of CREB Ser-133 phosphorylation and CBP/p300 recruitment. GC signaling through CD40 or the BCR, which activates pCREB-dependent genes, caused TORC2 phosphorylation, cytosolic emigration, and TCL1 repression. Signaling via cAMP-inducible pathways inhibited TCL1 repression and reduced apoptosis, consistent with a prosurvival role for TCL1 before GC selection and supporting an initiating role for aberrant TCL1 expression during GC lymphomagenesis. Our data indicate that a novel CREB/TORC2 regulatory mode controls the normal program of GC gene activation and repression that promotes B cell development and circumvents oncogenic progression. Our results also reconcile a paradox in which signals that activate pCREB/CBP/p300 genes concurrently repress TCL1 to initiate its silencing.

  9. Activation and repression functions of an SR splicing regulator depend on exonic versus intronic-binding position.

    PubMed

    Shen, Manli; Mattox, William

    2012-01-01

    SR proteins and related factors play widespread roles in alternative pre-mRNA splicing and are known to promote splice site recognition through their Arg-Ser-rich effector domains. However, binding of SR regulators to some targets results in repression of splice sites through a distinct mechanism. Here, we investigate how activated and repressed targets of the Drosophila SR regulator Transformer2 elicit its differing effects on splicing. We find that, like activation, repression affects early steps in the recognition of splice sites and spliceosome assembly. Repositioning of regulatory elements reveals that Tra2 complexes that normally repress splicing from intronic positions activate splicing when located in an exon. Protein tethering experiments demonstrate that this position dependence is an intrinsic property of Tra2 and further show that repression and activation are mediated by separate effector domains of this protein. When other Drosophila SR factors (SF2 and Rbp1) that activate splicing from exonic positions were tethered intronically they failed to either activate or repress splicing. Interestingly, both activities of Tra2 favor the exonic identity of the RNA sequences that encompass its binding sites. This suggests a model in which these two opposite functions act in concert to define both the position and extent of alternatively spliced exons.

  10. Testis determination requires a specific FGFR2 isoform to repress FOXL2.

    PubMed

    Bagheri-Fam, Stefan; Bird, Anthony D; Zhao, Liang; Ryan, Janelle M; Yong, Meiyun; Wilhelm, Dagmar; Koopman, Peter; Eswarakumar, Jacob V; Harley, Vincent R

    2017-09-11

    Male sex determination in mammals relies on SRY-mediated up-regulation of SOX9 expression in XY gonads, whereas WNT/RSPO signalling and FOXL2 drive female sex determination in XX gonads. FGF9-signalling ensures sustained SOX9 expression through repression of one of the ovarian pathways (WNT signalling), while the significance of FGF-mediated repression of the FOXL2 pathway has not been studied. Previously, we demonstrated that FGFR2 is the receptor for FGF9 in the XY gonad. Whether a specific isoform (FGFR2b or FGFR2c) is required was puzzling. Here, we show that FGFR2c is required for male sex determination. Initially, in developing mouse embryos at 12.5-13.5 days post coitum (dpc), XY Fgfr2c-/- gonads appear ovotestes, with SOX9 and FOXL2 expression predominantly localised to the posterior and anterior gonadal poles, respectively. However by 15.5dpc, XY Fgfr2c-/- gonads show complete male-to-female sex reversal, evident by the lack of SOX9 and ectopic expression of FOXL2 throughout the gonads. Furthermore, ablation of the Foxl2 gene leads to partial or complete rescue of gonadal sex reversal in XY Fgfr2c-/- mice. Together with previous findings, our data suggest that testis determination involves FGFR2c-mediated repression of both the WNT4- and FOXL2-driven ovarian determining pathways. Copyright © 2017 Endocrine Society.

  11. 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.

  12. Identification of a Potent Apoptotic Peptide Produced by Fibroblasts; Studies Towards the Design of a Novel Agent for Breast Cancer Therapy

    DTIC Science & Technology

    2007-09-05

    M., and Evans, R. M. (1995). 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma . Cell 83, 803-12...from apoptosis. This suggests that 15d-PGJ2 inhibits the production but not the activity of the apoptotic peptide. 15d-PGJ2 is a potent agonist for...on cellular proteins [20]. Therefore, we tested whether or not CAY10410, a 15d-PGJ2 analog that is a PPARγ agonist but cannot form adducts with

  13. Increased generation of cyclopentenone prostaglandins after brain ischemia and their role in aggregation of ubiquitinated proteins in neurons

    PubMed Central

    Liu, Hao; Li, Wenjin; Ahmad, Muzamil; Rose, Marie E.; Miller, Tricia M.; Yu, Mei; Chen, Jie; Pascoe, Jordan L.; Poloyac, Samuel M.; Hickey, Robert W.; Graham, Steven H.

    2013-01-01

    The cyclopentenone prostaglandin (CyPG) J2 series, including prostaglandin J2 (PGJ2), Δ12-PGJ2 and 15-deoxy-Δ12, 14 -prostaglandin J2 (15d-PGJ2), are active metabolites of PGD2, exerting multiple effects on neuronal function. However, the physiologic relevance of these effects remains uncertain as brain concentrations of CyPGs have not been precisely determined. In this study, we found that free PGD2 and the J2 series CyPGs (PGJ2, Δ12-PGJ2 and 15d-PGJ2) were increased in post-ischemic rat brain as detected by UPLC-MS/MS with 15d-PGJ2 being the most abundant CyPG. These increases were attenuated by pre-treating with the cyclooxygenase inhibitor piroxicam. Next, effects of chronic exposure to 15d-PGJ2 were examined by treating primary neurons with 15d-PGJ2, CAY10410 (a 15d-PGJ2 analog lacking the cyclopentenone ring structure), or vehicle for 24 h to 96 h. Because we found that the concentration of free 15d-PGJ2 decreased rapidly in cell culture medium, freshly prepared medium containing 15d-PGJ2, CAY10410 or vehicle was changed twice daily to maintain steady extracellular concentrations. Incubation with 2.5 μM 15d-PGJ2, but not CAY10410, increased neuronal cell death without induction of caspase-3 or PARP cleavage, consistent with a primarily necrotic mechanism for 15d-PGJ2-induced cell death which was further supported by TUNEL assay results. Ubiquitinated protein accumulation and aggregation was observed after 96 h 15d-PGJ2 incubation, accompanied by compromised 20S proteasome activity. Unlike another proteasome inhibitor, MG132, 15d-PGJ2 treatment did not activate autophagy or induce aggresome formation. Therefore, the cumulative cytotoxic effects of increased generation of CyPGs after stroke may contribute to delayed post-ischemic neuronal injury. PMID:23355003

  14. Bile Acids Function Synergistically To Repress Invasion Gene Expression in Salmonella by Destabilizing the Invasion Regulator HilD.

    PubMed

    Eade, Colleen R; Hung, Chien-Che; Bullard, Brian; Gonzalez-Escobedo, Geoffrey; Gunn, John S; Altier, Craig

    2016-08-01

    Salmonella spp. are carried by and can acutely infect agricultural animals and humans. After ingestion, salmonellae traverse the upper digestive tract and initiate tissue invasion of the distal ileum, a virulence process carried out by the type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1). Salmonellae coordinate SPI-1 expression with anatomical location via environmental cues, one of which is bile, a complex digestive fluid that causes potent repression of SPI-1 genes. The individual components of bile responsible for SPI-1 repression have not been previously characterized, nor have the bacterial signaling processes that modulate their effects been determined. Here, we characterize the mechanism by which bile represses SPI-1 expression. Individual bile acids exhibit repressive activity on SPI-1-regulated genes that requires neither passive diffusion nor OmpF-mediated entry. By using genetic methods, the effects of bile and bile acids were shown to require the invasion gene transcriptional activator hilD and to function independently of known upstream signaling pathways. Protein analysis techniques showed that SPI-1 repression by bile acids is mediated by posttranslational destabilization of HilD. Finally, we found that bile acids function synergistically to achieve the overall repressive activity of bile. These studies demonstrate a common mechanism by which diverse environmental cues (e.g., certain short-chain fatty acids and bile acids) inhibit SPI-1 expression. These data provide information relevant to Salmonella pathogenesis during acute infection in the intestine and during chronic infection of the gallbladder and inform the basis for development of therapeutics to inhibit invasion as a means of repressing Salmonella pathogenicity.

  15. Genotoxic properties of cyclopentenone prostaglandins and the onset of glutathione depletion.

    PubMed

    Solecki, Gergely Morten; Groh, Isabel Anna Maria; Kajzar, Julia; Haushofer, Carolin; Scherhag, Anne; Schrenk, Dieter; Esselen, Melanie

    2013-02-18

    Prostaglandins are endogenous mediators formed from arachidonic acid by cyclooxygenases and prostaglandin synthases during inflammatory processes. The five-membered ring can be dehydrated, and α,β-unsaturated cyclopentenone PGs (cyPGs) are generated. Recent studies have been focused on their potential pharmacological use against inflammation and cancer. However, little is known so far about possible adverse health effects of cyPGs. We addressed the question whether selected cyPGs at a concentration range of 0.1-10 μM exhibit mutagenic and genotoxic properties in the hamster lung fibroblast V79 cell line and whether these effects are accompanied by a depletion of intracellular glutathione (GSH). The cyPGs 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) and prostaglandin A2 (PGA2) significantly induced DNA damage in V79 cells after 1 h of incubation. Furthermore, a more pronounced increase in formamidopyrimidine-DNA glycosylase (FPG) sensitive sites, indicative of oxidative DNA-damage, was observed. The findings on DNA-damaging properties were supported by our results that 15dPGJ(2) acts as an aneugenic agent which induces the amount of kinetochore positive micronuclei associated with an increase of apoptosis. The strong potency of cyPGs to rapidly bind GSH measured in a chemical assay and to significantly reduce the GSH level after only 1 h of incubation may contribute to the observed oxidative DNA strand breaks, whereas directly induced oxidative stress via reactive oxygen species could be excluded. However, after an extended incubation time of 24 h no genotoxicity could be measured, this may contribute to the lack of mutagenicity in the hypoxanthine phosphorybosyltransferase (HPRT) assay. In conclusion, potential in vitro genotoxicity of cyPG and a strong impact on GSH homeostasis have been demonstrated, which may be involved in carcinogenesis mediated by chronic inflammation.

  16. Stimulation of CD36 and the key effector of reverse cholesterol transport ATP-binding cassette A1 in monocytoid cells by niacin.

    PubMed

    Rubic, Tina; Trottmann, Matthias; Lorenz, Reinhard L

    2004-02-01

    Niacin, the first lipid lowering drug shown to improve survival after myocardial infarction, decreases LDL and increases HDL cholesterol levels. These effects cannot fully be explained by its suspected mechanism of action, inhibition of lipolysis and hepatic VLDL synthesis. Niacin has also been shown to interfere with the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and massively stimulate prostaglandin D2 (PGD2) formation. The major metabolite of PGD2, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), was recently identified as the most potent endogenous PPARgamma activator. We, therefore, studied the effects of niacin on the PPARgamma- and cAMP-dependent expression of receptors promoting reverse cholesterol transport. The transcription of PPARgamma-, HDL-, LDL- and scavenger-receptors and the sterol exporter ABCA1, were measured by quantitative RT-PCR and cellular cholesterol efflux and PPARgamma activation studied in macrophage and hepatocyte models. Niacin stimulated the translocation of PPARgamma and the transcription of PPARgamma, CD36 and ABCA1 in monocytoid cells, whereas the LDL-receptor (LDL-R) was unchanged. Thereby niacin enhanced HDL-mediated cholesterol efflux from the cells resulting in a reduced cellular cholesterol content. The niacin effect on CD36 but not on ABCA1 was prevented by cyclooxygenase inhibition, whereas the niacin effect on ABCA1 but not on CD36 was prevented by PKA inhibition, suggesting mediation by the 15d-PGJ2/PPARgamma and the cAMP/PKA pathways, respectively. These new actions of niacin on several key effectors of reverse cholesterol transport out of the vessel wall provide a rational to expect regression of atherosclerosis and test the combination of niacin with statins for an overadditive clinical benefit.

  17. 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

  18. MeCP2 Repression of G9a in Regulation of Pain and Morphine Reward

    PubMed Central

    Zhang, Zhi; Tao, Wenjuan; Hou, Yuan-Yuan; Wang, Wei; Kenny, Paul J.

    2014-01-01

    Opioids are commonly used for pain relief, but their strong rewarding effects drive opioid misuse and abuse. How pain affects the liability of opioid abuse is unknown at present. In this study, we identified an epigenetic regulating cascade activated by both pain and the opioid morphine. Both persistent pain and repeated morphine upregulated the transcriptional regulator MeCP2 in mouse central nucleus of the amygdala (CeA). Chromatin immunoprecipitation analysis revealed that MeCP2 bound to and repressed the transcriptional repressor histone dimethyltransferase G9a, reducing G9a-catalyzed repressive mark H3K9me2 in CeA. Repression of G9a activity increased expression of brain-derived neurotrophic factor (BDNF). Behaviorally, persistent inflammatory pain increased the sensitivity to acquiring morphine-induced, reward-related behavior of conditioned place preference in mice. Local viral vector-mediated MeCP2 overexpression, Cre-induced G9a knockdown, and CeA application of BDNF mimicked, whereas MeCP2 knockdown inhibited, the pain effect. These results suggest that MeCP2 directly represses G9a as a shared mechanism in central amygdala for regulation of emotional responses to pain and opioid reward, and for their behavioral interaction. PMID:24990928

  19. OprD Repression upon Metal Treatment Requires the RNA Chaperone Hfq in Pseudomonas aeruginosa

    PubMed Central

    Ducret, Verena; Gonzalez, Manuel R.; Scrignari, Tiziana; Perron, Karl

    2016-01-01

    The metal-specific CzcRS two-component system in Pseudomonas aeruginosa is involved in the repression of the OprD porin, causing in turn carbapenem antibiotic resistance in the presence of high zinc concentration. It has also been shown that CzcR is able to directly regulate the expression of multiple genes including virulence factors. CzcR is therefore an important regulator connecting (i) metal response, (ii) pathogenicity and (iii) antibiotic resistance in P. aeruginosa. Recent data have suggested that other regulators could negatively control oprD expression in the presence of zinc. Here we show that the RNA chaperone Hfq is a key factor acting independently of CzcR for the repression of oprD upon Zn treatment. Additionally, we found that an Hfq-dependent mechanism is necessary for the localization of CzcR to the oprD promoter, mediating oprD transcriptional repression. Furthermore, in the presence of Cu, CopR, the transcriptional regulator of the CopRS two-component system also requires Hfq for oprD repression. Altogether, these results suggest important roles for this RNA chaperone in the context of environment-sensing and antibiotic resistance in P. aeruginosa. PMID:27706108

  20. Somatic sex determination in Caenorhabditis elegans is modulated by SUP-26 repression of tra-2 translation

    PubMed Central

    Mapes, James; Chen, Jeng-Ting; Yu, Jau-Song; Xue, Ding

    2010-01-01

    Translational repression mediated by RNA-binding proteins or micro RNAs has emerged as a major regulatory mechanism for fine-tuning important biological processes. In Caenorhabditis elegans, translational repression of the key sex-determination gene tra-2 (tra, transformer) is controlled by a 28-nucleotide repeat element, the TRA-2/GLI element (TGE), located in its 3′ untranslated region (UTR). Mutations that disrupt TGE or the germline-specific TGE-binding factor GLD-1 increase TRA-2 protein expression and inhibit sperm production in hermaphrodites. Here we report the characterization of the sup-26 gene, which regulates sex determination in the soma and encodes an RNA recognition motif (RRM)-containing protein. We show that SUP-26 regulates the level of the TRA-2 protein through TGE in vivo and binds directly to TGE in vitro through its RRM domain. Interestingly, SUP-26 associates with poly(A)-binding protein 1 (PAB-1) in vivo and may repress tra-2 expression by inhibiting the translation-stimulating activity of PAB-1. Taken together, our results provide further insight into how mRNA-binding factors repress translation and modulate sexual development in different tissues of C. elegans. PMID:20921392

  1. Somatic sex determination in Caenorhabditis elegans is modulated by SUP-26 repression of tra-2 translation.

    PubMed

    Mapes, James; Chen, Jeng-Ting; Yu, Jau-Song; Xue, Ding

    2010-10-19

    Translational repression mediated by RNA-binding proteins or micro RNAs has emerged as a major regulatory mechanism for fine-tuning important biological processes. In Caenorhabditis elegans, translational repression of the key sex-determination gene tra-2 (tra, transformer) is controlled by a 28-nucleotide repeat element, the TRA-2/GLI element (TGE), located in its 3' untranslated region (UTR). Mutations that disrupt TGE or the germline-specific TGE-binding factor GLD-1 increase TRA-2 protein expression and inhibit sperm production in hermaphrodites. Here we report the characterization of the sup-26 gene, which regulates sex determination in the soma and encodes an RNA recognition motif (RRM)-containing protein. We show that SUP-26 regulates the level of the TRA-2 protein through TGE in vivo and binds directly to TGE in vitro through its RRM domain. Interestingly, SUP-26 associates with poly(A)-binding protein 1 (PAB-1) in vivo and may repress tra-2 expression by inhibiting the translation-stimulating activity of PAB-1. Taken together, our results provide further insight into how mRNA-binding factors repress translation and modulate sexual development in different tissues of C. elegans.

  2. Repression activity of Tailless on h 1 and eve 1 pair-rule stripes.

    PubMed

    Andrioli, Luiz Paulo; Dos Santos, Wesley Silva; Aguiar, Francisco Dos Santos; Digiampietri, Luciano Antonio

    2017-04-01

    We investigated the hypothesis that several transcriptional repressors are necessary to set the boundaries of anterior pair-rule stripes in Drosophila. Specifically, we tested whether Tailless (Tll) is part of a repression mechanism that correctly sets the anterior boundaries of hairy 1 (h 1) and even-skipped 1 (eve 1) stripes. Single mutant tll embryos displayed subtle deviations from the normal positions of h 1 and eve 1 stripes. Moreover, we observed stronger stripe deviations in embryos lacking both Tll and Sloppy-paired 1 (Slp 1), a common repressor for anterior pair-rule stripes. Using h 1 and eve 1 reporter constructs in the genetic assays, we provided further evidence that interference with normal mechanisms of stripe expression is mediated by Tll repression. Indeed, Tll represses both h 1 and eve 1 reporter stripes when misexpressed. Investigating the expression of other anterior gap genes in different genetic backgrounds and in the misexpression assays strengthened Tll direct repression in the regulation of h 1 and eve 1. Our results are consistent with tll being a newly-identified component of a combinatorial network of repressor genes that control pair-rule stripe formation in the anterior blastoderm of Drosophila. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. MeCP2 repression of G9a in regulation of pain and morphine reward.

    PubMed

    Zhang, Zhi; Tao, Wenjuan; Hou, Yuan-Yuan; Wang, Wei; Kenny, Paul J; Pan, Zhizhong Z

    2014-07-02

    Opioids are commonly used for pain relief, but their strong rewarding effects drive opioid misuse and abuse. How pain affects the liability of opioid abuse is unknown at present. In this study, we identified an epigenetic regulating cascade activated by both pain and the opioid morphine. Both persistent pain and repeated morphine upregulated the transcriptional regulator MeCP2 in mouse central nucleus of the amygdala (CeA). Chromatin immunoprecipitation analysis revealed that MeCP2 bound to and repressed the transcriptional repressor histone dimethyltransferase G9a, reducing G9a-catalyzed repressive mark H3K9me2 in CeA. Repression of G9a activity increased expression of brain-derived neurotrophic factor (BDNF). Behaviorally, persistent inflammatory pain increased the sensitivity to acquiring morphine-induced, reward-related behavior of conditioned place preference in mice. Local viral vector-mediated MeCP2 overexpression, Cre-induced G9a knockdown, and CeA application of BDNF mimicked, whereas MeCP2 knockdown inhibited, the pain effect. These results suggest that MeCP2 directly represses G9a as a shared mechanism in central amygdala for regulation of emotional responses to pain and opioid reward, and for their behavioral interaction.

  4. 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

  5. Bmp signaling represses Vegfa to promote outflow tract cushion development.

    PubMed

    Bai, Yan; Wang, Jun; Morikawa, Yuka; Bonilla-Claudio, Margarita; Klysik, Elzbieta; Martin, James F

    2013-08-01

    Congenital heart disease (CHD) is a devastating anomaly that affects ∼1% of live births. Defects of the outflow tract (OFT) make up a large percentage of human CHD. We investigated Bmp signaling in mouse OFT development by conditionally deleting both Bmp4 and Bmp7 in the second heart field (SHF). SHF Bmp4/7 deficiency resulted in defective epithelial to mesenchymal transition (EMT) and reduced cardiac neural crest ingress, with resultant persistent truncus arteriosus. Using a candidate gene approach, we found that Vegfa was upregulated in the Bmp4/7 mutant hearts. To determine if Vegfa is a downstream Bmp effector during EMT, we examined whether Vegfa is transcriptionally regulated by the Bmp receptor-regulated Smad. Our findings indicate that Smad directly binds to Vegfa chromatin and represses Vegfa transcriptional activity. We also found that Vegfa is a direct target for the miR-17-92 cluster, which is also regulated by Bmp signaling in the SHF. Deletion of miR-17-92 reveals similar phenotypes to Bmp4/7 SHF deletion. To directly address the function of Vegfa repression in Bmp-mediated EMT, we performed ex vivo explant cultures from Bmp4/7 and miR-17-92 mutant hearts. EMT was defective in explants from the Bmp4/7 double conditional knockout (dCKO; Mef2c-Cre;Bmp4/7(f/f)) and miR-17-92 null. By antagonizing Vegfa activity in explants, EMT was rescued in Bmp4/7 dCKO and miR-17-92 null culture. Moreover, overexpression of miR-17-92 partially suppressed the EMT defect in Bmp4/7 mutant embryos. Our study reveals that Vegfa levels in the OFT are tightly controlled by Smad- and microRNA-dependent pathways to modulate OFT development.

  6. Selective repression of SINE transcription by RNA polymerase III.

    PubMed

    Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J; Cairns, Bradley R; White, Robert J

    2015-01-01

    A million copies of the Alu short interspersed nuclear element (SINE) are scattered throughout the human genome, providing ∼11% of our total DNA. SINEs spread by retrotransposition, using a transcript generated by RNA polymerase (pol) III from an internal promoter. Levels of these pol III-dependent Alu transcripts are far lower than might be expected from the abundance of the template. This was believed to reflect transcriptional suppression through DNA methylation, denying pol III access to most SINEs through chromatin-mediated effects. Contrary to expectations, our recent study found no evidence that methylation of SINE DNA reduces its occupancy or expression by pol III. However, histone H3 associated with SINEs is prominently methylated on lysine 9, a mark that correlates with transcriptional silencing. The SUV39 methyltransferases that deposit this mark can be found at many SINEs. Furthermore, a selective inhibitor of SUV39 stimulates pol III recruitment to these loci, as well as SINE expression. These data suggest that methylation of histone H3 rather than DNA may mediate repression of SINE transcription by pol III, at least under the conditions we studied.

  7. The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.

    PubMed

    Oeste, Clara L; Díez-Dacal, Beatriz; Bray, Francesca; García de Lacoba, Mario; de la Torre, Beatriz G; Andreu, David; Ruiz-Sánchez, Antonio J; Pérez-Inestrosa, Ezequiel; García-Domínguez, Carlota A; Rojas, José M; Pérez-Sala, Dolores

    2011-01-06

    Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2) selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2). Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

  8. 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

  9. Intrathecal rosiglitazone acts at peroxisome proliferator-activated receptor γ to rapidly inhibit neuropathic pain in rats

    PubMed Central

    Churi, Sajay B.; Abdel-Aleem, Omar S.; Tumber, Kiranjeet K.; Scuderi-Porter, Heather; Taylor, Bradley K.

    2008-01-01

    We first demonstrate the transcription, expression, and DNA binding properties of the PPARγ subtype of the peroxisome proliferator-activated nuclear receptor family to the spinal cord with real time PCR, western blot, and electrophoretic mobility shift assay. To test the hypothesis that activation of spinal PPARγ decreases nerve injury-induced allodynia, we intrathecally administered PPARγ agonists and/or antagonists in rats following transection of the tibial and common peroneal branches of the sciatic nerve. Single injection of either a natural (15-deoxy-prostaglandin J2, 15d-PGJ2) or synthetic (rosiglitazone) PPARγ agonist dose-dependently decreased mechanical and cold hypersensitivity. These effects were maximal at a dose of 100μg and peaked at ~60 min after injection, a rapid time course suggestive of transcription-independent mechanisms of action. Concurrent administration of a PPARγ antagonist (bisphenol A diglycidyl ether, BADGE) reversed the effects of 15d-PGJ2 and rosiglitazone, further indicating a receptor-mediated effect. In animals without nerve injury, rosiglitazone did not alter motor coordination, von Frey threshold, or withdrawal response to a cool stimulus. Intraperitoneal and intracerebroventricular administration of PPARγ agonists (100μg) did not decrease mechanical and cold hypersensitivity, arguing against effects subsequent to diffusion from the intrathecal space. We conclude that ligand-induced activation of spinal PPARγ rapidly reverses nerve injury-induced mechanical allodynia. New or currently-available drugs targeted at spinal PPARγ may yield important therapeutic effects for the management of neuropathic pain. PERSPECTIVE PPARγ receptor agonists such as rosiglitazone and pioglitazone remain FDA approved as insulin sensitizers. We demonstrate PPARγ expression in the spinal cord, and report that activation of these receptors inhibits allodynia. BBB-permeant PPARγ agonists may yield important therapeutic effects for the

  10. Pro-/anti-inflammatory dysregulation in patients with first episode of psychosis: toward an integrative inflammatory hypothesis of schizophrenia.

    PubMed

    García-Bueno, Borja; Bioque, Miquel; Mac-Dowell, Karina S; Barcones, M Fe; Martínez-Cengotitabengoa, Monica; Pina-Camacho, Laura; Rodríguez-Jiménez, Roberto; Sáiz, Pilar A; Castro, Carmen; Lafuente, Amalia; Santabárbara, Javier; González-Pinto, Ana; Parellada, Mara; Rubio, Gabriel; García-Portilla, M Paz; Micó, Juan A; Bernardo, Miguel; Leza, Juan C

    2014-03-01

    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. 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. 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. 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.

  11. Regulation of retinoic acid receptor beta expression by peroxisome proliferator-activated receptor gamma ligands in cancer cells.

    PubMed

    James, Sharon Y; Lin, Feng; Kolluri, Siva Kumar; Dawson, Marcia I; Zhang, Xiao-kun

    2003-07-01

    The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor family member that can form a heterodimeric complex with retinoid X receptor (RXR) and initiate transcription of target genes. In this study, we have examined the effects of the PPAR gamma ligand ciglitazone and the RXR ligand SR11237 on growth and induction of retinoic acid receptor (RAR) beta expression in breast and lung cancer cells. Our results demonstrated that ciglitazone and SR11237 cooperatively inhibited the growth of ZR-75-1 and T-47D breast cancer and Calu-6 lung cancer cells. Gel shift analysis indicated that PPAR gamma, in the presence of RXR, formed a strong complex with a retinoic acid response element (beta retinoic acid response element) in the RAR beta promoter. In reporter gene assays, RXR ligands and ciglitazone, but not the PPAR gamma ligand 15d-PGJ(2), cooperatively promoted the transcriptional activity of the beta retinoic acid response element. Ciglitazone, but not 15d-PGJ(2), strongly induced RAR beta expression in human breast and lung cancer cell lines when used together with SR11237. The induction of RAR beta expression by the ciglitazone and SR11237 combination was diminished by a PPAR gamma-selective antagonist, bisphenol A diglycidyl ether. All-trans-retinoic acid or the combination of ciglitazone and SR11237 was able to induce RAR beta in all-trans-retinoic acid-resistant MDA-MB-231 breast cancer cells only when the orphan receptor chick ovalbumin upstream promoter transcription factor was expressed, or in the presence of the histone deacetylase inhibitor trichostatin A. These studies indicate the existence of a novel RAR beta-mediated signaling pathway of PPAR gamma action, which may provide a molecular basis for developing novel therapies involving RXR and PPAR gamma ligands in potentiating antitumor responses.

  12. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Induction and Repression of Amidase Enzymes in Aspergillus nidulans

    PubMed Central

    Hynes, M. J.

    1970-01-01

    Aspergillus nidulans can grow on acetamide as both a carbon and nitrogen source and can also grow on formamide as a nitrogen source. Two distinct enzymes, an acetamidase and a formamidase, are produced. The control of the synthesis of these two enzymes in a wild-type strain was investigated. The formamidase is induced by acetamide and formamide and repressed by ammonia. The acetamidase is induced by formamide and acetamide, repressed by carbon metabolites derived from glucose and acetate, and repressed by ammonia. Repression of the acetamidase by ammonia depends on the carbon source; growth on glucose but not on acetate or acetamide allows repression to occur. The pattern of acetamidase repression is compared with that of histidine catabolic enzymes in various bacteria. PMID:5432013

  14. BRCA1 and c-Myc associate to transcriptionally repress psoriasin, a DNA damage-inducible gene.

    PubMed

    Kennedy, Richard D; Gorski, Julia J; Quinn, Jennifer E; Stewart, Gail E; James, Colin R; Moore, Stephen; Mulligan, Karl; Emberley, Ethan D; Lioe, Tong F; Morrison, Patrick J; Mullan, Paul B; Reid, George; Johnston, Patrick G; Watson, Peter H; Harkin, D Paul

    2005-11-15

    Evidence is accumulating to suggest that some of the diverse functions associated with BRCA1 may relate to its ability to transcriptionally regulate key downstream target genes. Here, we identify S100A7 (psoriasin), S100A8, and S100A9, members of the S100A family of calcium-binding proteins, as novel BRCA1-repressed targets. We show that functional BRCA1 is required for repression of these family members and that a BRCA1 disease-associated mutation abrogates BRCA1-mediated repression of psoriasin. Furthermore, we show that BRCA1 and c-Myc form a complex on the psoriasin promoter and that BRCA1-mediated repression of psoriasin is dependent on functional c-Myc. Finally, we show that psoriasin expression is induced by the topoisomerase IIalpha poison, etoposide, in the absence of functional BRCA1 and increased psoriasin expression enhances cellular sensitivity to this chemotherapeutic agent. Therefore, we identified a novel transcriptional mechanism that is likely to contribute to BRCA1-mediated resistance to etoposide.

  15. Protein inhibitor of activated STAT 4 (PIAS4) regulates pro-inflammatory transcription in hepatocytes by repressing SIRT1.

    PubMed

    Sun, Lina; Fan, Zhiwen; Chen, Junliang; Tian, Wenfang; Li, Min; Xu, Huihui; Wu, Xiaoyan; Fang, Mingming; Xia, Jun; Xu, Yong

    2016-07-12

    Excessive nutrition promotes the pathogenesis of non-alc