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Sample records for active gene transcription

  1. Transcriptional activation of ribosomal RNA genes during compensatory renal hypertrophy

    SciTech Connect

    Ouellette, A.J.; Moonka, R.; Zelenetz, A.; Malt, R.A.

    1986-05-01

    The overall rate of rDNA transcription increases by 50% during the first 24 hours of compensatory renal hypertrophy in the mouse. To study mechanisms of ribosome accumulation after uninephrectomy, transcription rates were measured in isolated kidneys by transcriptional runoff. /sup 32/P-labeled nascent transcripts were hybridized to blots containing linearized, denatured cloned rDNA, and hybridization was quantitated autoradiographically and by direct counting. Overall transcriptional activity of rDNA was increased by 30% above control levels at 6 hrs after nephrectomy and by 50% at 12, 18, and 24 hrs after operation. Hybridizing RNA was insensitive to inhibiby alpha-amanitin, and no hybridization was detected to vector DNA. Thus, accelerated rDNA transcription is one regulatory element in the accretion of ribosomes in renal growth, and the regulatory event is an early event. Mechanisms of activation may include enhanced transcription of active genes or induction of inactive DNA.

  2. The murine Sry gene encodes a nuclear transcriptional activator

    SciTech Connect

    Dubin, R.A.; Ostrer, H.

    1994-09-01

    The Sry gene functions as a genetic switch in gonadal ridge initiating testis determination. The murine Sry and human SRY open reading frames (ORF) share a conserved 79 amino acid motif, the HMG-box, that binds DNA. Outside this region the two genes share no additional homology. These studies were undertaken to determine whether the Sry/SRY genes encode nuclear transcriptional regulators. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and murine SRY ORFs contain a nuclear localization signal. The murine Sry HMG-box selectively binds the sequence NACAAT in vitro when presented with a random pool of oligonucleotides and binds AACAAT with the highest affinity. The murine Sry ORF, when expressed in HeLa cells, activates transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was observed for a GAL4-responsive gene when the murine Sry ORF was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a C-terminal glutamine/histidine-rich domain. In addition, LexA-Sry fusion genes activated a LexA-responsive gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and mouse SRY ORFs encode nuclear, DNA-binding proteins, and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

  3. Human DJ-1-specific Transcriptional Activation of Tyrosine Hydroxylase Gene*

    PubMed Central

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M. M.

    2010-01-01

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-l-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice. PMID:20938049

  4. Human DJ-1-specific transcriptional activation of tyrosine hydroxylase gene.

    PubMed

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M M

    2010-12-17

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-L-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice.

  5. Transcriptional activation of virulence genes of Rhizobium etli.

    PubMed

    Wang, Luyao; Lacroix, Benoît; Guo, Jianhua; Citovsky, Vitaly

    2017-01-09

    Recently, Rhizobium etli has emerged, in addition to Agrobacterium spp., as a prokaryotic species that encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we showed that the signal phenolic molecule acetosyringone (AS) induced R. etli vir gene expression both in R. etli and in A. tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter T-DNA. Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium, but not from non-host plants. Early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside of the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a versus pTiC58 of A. tumefaciens IMPORTANCE: The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into plant cell genome. In this study, we explore the transcriptional regulation and induction of virulence genes in R. etli and show similarities and differences

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

    EPA Science Inventory

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Thiel, Gerald; Rössler, Oliver G

    2017-03-01

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

  9. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products.

    PubMed

    Green, M R; Treisman, R; Maniatis, T

    1983-11-01

    When the human beta-globin gene is transfected into Hela cells, no beta-globin RNA is detected unless the gene is linked to a viral transcription enhancer. In this paper we show that trans-acting adenovirus and herpesvirus (pseudorabies) transcriptional regulatory proteins can circumvent this enhancer requirement for detectable beta-globin transcription in transient expression assays. The viral gene products can be provided by constitutively expressed, integrated viral genes in established cell lines, by viral infection of permissive cells, or by transfection of cells with bacterial plasmids carrying the viral immediate-early genes. These results demonstrate the utility of transient expression assays for studying regulatory mechanisms involving trans-acting factors. Analysis of beta-globin promoter mutants indicates that between 75 and 128 bp of sequence 5' to the mRNA cap site is required for enhancer-dependent transcription in Hela cells. In contrast, beta-globin transcription in the presence of viral immediate-early gene products requires only 36 bp of 5'-flanking sequence, which includes the TATA box. Thus both cis and trans-acting viral factors activate beta-globin gene transcription in transient expression experiments, but the mechanisms by which they act appear to be fundamentally different.

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

    EPA Science Inventory

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

  11. Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

    NASA Technical Reports Server (NTRS)

    Morrow, Maureen A.

    2003-01-01

    Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

  12. Transcriptional Activation of Inflammatory Genes: Mechanistic Insight into Selectivity and Diversity

    PubMed Central

    Ahmed, Afsar U.; Williams, Bryan R. G.; Hannigan, Gregory E.

    2015-01-01

    Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding. PMID:26569329

  13. Activation of tissue plasminogen activator gene transcription by Neovastat, a multifunctional antiangiogenic agent.

    PubMed

    Gingras, Denis; Nyalendo, Carine; Di Tomasso, Geneviève; Annabi, Borhane; Béliveau, Richard

    2004-07-16

    We recently reported that Neovastat, an antiangiogenic drug that is currently undergoing Phase III clinical trials for the treatment of non-small cell lung cancer, may inhibit angiogenesis through an increase in tPA activity. Here, we show that Neovastat also stimulates tPA gene transcription in endothelial cells, in a TNFalpha-like manner. RT-PCR analysis and gene reporter assays using the human tPA promoter indicated that upregulation of the tPA gene transcription by both Neovastat and TNFalpha was correlated with the phosphorylation of JNK1/2 and of IkappaB and that SP600125 and BAY11-7082, inhibitors of JNK and IkappaK, respectively, inhibit the increase of tPA gene transcription induced by Neovastat and TNFalpha. These results suggest that Neovastat induces tPA gene transcription through activation of the JNK and NFkappaB signaling pathways, leading to an increase of tPA secretion by endothelial cells. This may lead to the localized destruction of the fibrin provisional matrix that is necessary for neovessel formation and thus contribute to the reported antiangiogenic properties of this compound.

  14. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    PubMed

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms.

  15. Adenovirus E1A protein activates transcription of the E1A gene subsequent to transcription complex formation.

    PubMed Central

    Schaack, J; Logan, J; Vakalopoulou, E; Shenk, T

    1991-01-01

    The mechanism of transcriptional activation of the adenovirus E1A and E3 genes by E1A protein during infection was examined by using transcription-competition assays. Infection of HeLa cells with one virus led to inhibition of mRNA accumulation from a superinfecting virus. Synthesis of the E1A 289R protein by the first virus to infect reduced inhibition of transcription of the superinfecting virus, indicating that the E1A 289R protein was limiting for E1A-activated transcription. Infection with an E1A- virus, followed 6 h later by superinfection with a wild-type virus, led to preferential transcriptional activation of the E1A gene of the first virus, suggesting that a host transcription component(s) stably associated with the E1A promoter in the absence of E1A protein and that this complex was the substrate for transcriptional activation by E1A protein. The limiting host transcription component(s) bound to the E1A promoter to form a complex with a half-life greater than 24 h in the absence of E1A 289R protein, as demonstrated in a challenge assay with a large excess of superinfecting virus. In the presence of the E1A 289R protein, the E1A gene of the superinfecting virus was gradually activated with a reduction in E1A mRNA accumulation from the first virus. The kinetics of the activation suggest that this was due to an indirect effect rather than to destabilization of stable transcription complexes by the 289R protein. Images PMID:1825853

  16. SUMO functions in constitutive transcription and during activation of inducible genes in yeast.

    PubMed

    Rosonina, Emanuel; Duncan, Sarah M; Manley, James L

    2010-06-15

    Transcription factors represent one of the largest groups of proteins regulated by SUMO (small ubiquitin-like modifier) modification, and their sumoylation is usually associated with transcriptional repression. To investigate whether sumoylation plays a general role in regulating transcription in yeast, we determined the occupancy of sumoylated proteins at a variety of genes by chromatin immunoprecipitation (ChIP) using an antibody that recognizes the yeast SUMO peptide. Surprisingly, we detected sumoylated proteins at all constitutively transcribed genes tested but not at repressed genes. Ubc9, the SUMO conjugation enzyme, was not present on these genes, but its inactivation reduced SUMO at the constitutive promoters and modestly decreased RNA polymerase II levels. In contrast, activation of the inducible GAL1, STL1, and ARG1 genes caused not only a striking accumulation of SUMO at all three promoter regions, but also recruitment of Ubc9, indicating that gene activation involves sumoylation of promoter-bound factors. However, Ubc9 inactivation, while reducing sumoylation at the induced promoters, paradoxically resulted in increased transcription. Providing an explanation for this, the reduced sumoylation impaired the cell's ability to appropriately shut off transcription of the induced ARG1 gene, indicating that SUMO can facilitate transcriptional silencing. Our findings thus establish unexpected roles for sumoylation in both constitutive and activated transcription, and provide a novel mechanism for regulating gene expression.

  17. Aurora kinase B activity is modulated by thyroid hormone during transcriptional activation of pituitary genes.

    PubMed

    Tardáguila, Manuel; González-Gugel, Elena; Sánchez-Pacheco, Aurora

    2011-03-01

    Covalent histone modifications clearly play an essential role in ligand-dependent transcriptional regulation by nuclear receptors. One of the predominant mechanisms used by nuclear receptors to activate or repress target-gene transcription is the recruitment of coregulatory factors capable of covalently modify the amino terminal ends of histones. Here we show that the thyroid hormone (T3) produces a rapid increase in histone H3Ser10 phosphorylation (H3Ser10ph) concomitant to the rapid displacement of the heterochromatin protein 1β (HP1β) to the nuclear periphery. Moreover, we found that T3-mediated pituitary gene transcription is associated with an increase in H3Ser10ph. Interestingly, the Aurora kinase B inhibitor ZM443979 abolishes the effect of T3 on H3Ser10ph, blocks HP1β delocalization, and significantly reduces ligand-dependent transactivation. Similar effects were shown when Aurora kinase B expression was abrogated in small interfering RNA assays. In an effort to understand the underlying mechanism by which T3 increases H3Ser10ph, we demonstrate that liganded thyroid hormone receptor directly interacts with Aurora kinase B, increasing its kinase activity. Moreover, using chromatin immunoprecipitation assays, we have shown that Aurora kinase B participates of a mechanism that displaces HP1β from promoter region, thus preparing the chromatin for the transcriptional activation of T3 regulated genes. Our findings reveal a novel role for Aurora kinase B during transcriptional initiation in GO/G1, apart from its well-known mitotic activity.

  18. Partially phosphorylated Pho4 activates transcription of a subset of phosphate-responsive genes.

    PubMed

    Springer, Michael; Wykoff, Dennis D; Miller, Nicole; O'Shea, Erin K

    2003-11-01

    A cell's ability to generate different responses to different levels of stimulus is an important component of an adaptive environmental response. Transcriptional responses are frequently controlled by transcription factors regulated by phosphorylation. We demonstrate that differential phosphorylation of the budding yeast transcription factor Pho4 contributes to differential gene expression. When yeast cells are grown in high-phosphate growth medium, Pho4 is phosphorylated on four critical residues by the cyclin-CDK complex Pho80-Pho85 and is inactivated. When yeast cells are starved for phosphate, Pho4 is dephosphorylated and fully active. In intermediate-phosphate conditions, a form of Pho4 preferentially phosphorylated on one of the four sites accumulates and activates transcription of a subset of phosphate-responsive genes. This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression. Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80-Pho85, and one transcription factor, Pho4. Differential phosphorylation of Pho4 by Pho80-Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs.

  19. Partially Phosphorylated Pho4 Activates Transcription of a Subset of Phosphate-Responsive Genes

    PubMed Central

    Miller, Nicole

    2003-01-01

    A cell's ability to generate different responses to different levels of stimulus is an important component of an adaptive environmental response. Transcriptional responses are frequently controlled by transcription factors regulated by phosphorylation. We demonstrate that differential phosphorylation of the budding yeast transcription factor Pho4 contributes to differential gene expression. When yeast cells are grown in high-phosphate growth medium, Pho4 is phosphorylated on four critical residues by the cyclin–CDK complex Pho80–Pho85 and is inactivated. When yeast cells are starved for phosphate, Pho4 is dephosphorylated and fully active. In intermediate-phosphate conditions, a form of Pho4 preferentially phosphorylated on one of the four sites accumulates and activates transcription of a subset of phosphate-responsive genes. This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression. Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80–Pho85, and one transcription factor, Pho4. Differential phosphorylation of Pho4 by Pho80–Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs. PMID:14624238

  20. Patterns of Gene-Specific and Total Transcriptional Activity during the Plasmodium falciparum Intraerythrocytic Developmental Cycle ▿ †

    PubMed Central

    Sims, Jennifer S.; Militello, Kevin T.; Sims, Peter A.; Patel, Vishal P.; Kasper, Jacob M.; Wirth, Dyann F.

    2009-01-01

    The relationships among gene regulatory mechanisms in the malaria parasite Plasmodium falciparum throughout its asexual intraerythrocytic developmental cycle (IDC) remain poorly understood. To investigate the level and nature of transcriptional activity and its role in controlling gene expression during the IDC, we performed nuclear run-on on whole-transcriptome samples from time points throughout the IDC and found a peak in RNA polymerase II-dependent transcriptional activity related to both the number of nuclei per parasite and variable transcriptional activity per nucleus over time. These differential total transcriptional activity levels allowed the calculation of the absolute transcriptional activities of individual genes from gene-specific nuclear run-on hybridization data. For half of the genes analyzed, sense-strand transcriptional activity peaked at the same time point as total activity. The antisense strands of several genes were substantially transcribed. Comparison of the transcriptional activity of the sense strand of each gene to its steady-state RNA abundance across the time points assayed revealed both correlations and discrepancies, implying transcriptional and posttranscriptional regulation, respectively. Our results demonstrate that such comparisons can effectively indicate gene regulatory mechanisms in P. falciparum and suggest that genes with diverse transcriptional activity levels and patterns combine to produce total transcriptional activity levels tied to parasite development during the IDC. PMID:19151330

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

    PubMed

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

    2012-06-29

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

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

    PubMed

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

    1998-08-01

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

  3. Transcriptional Activity of rRNA Genes in Barley Cells after Mutagenic Treatment

    PubMed Central

    2016-01-01

    In the present study, the combination of the micronucleus test with analysis of the activity of the rRNA genes in mutagen-treated Hordeum vulgare (barley) by maleic hydrazide (MH) cells was performed. Simultaneously fluorescence in situ hybridization (FISH) with 25S rDNA as probes and an analysis of the transcriptional activity of 35S rRNA genes with silver staining were performed. The results showed that transcriptional activity is always maintained in the micronuclei although they are eliminated during the next cell cycle. The analysis of the transcriptional activity was extended to barley nuclei. MH influenced the fusion of the nucleoli in barley nuclei. The silver staining enabled detection of the nuclear bodies which arose after MH treatment. The results confirmed the usefulness of cytogenetic techniques in the characterization of micronuclei. Similar analyses can be now extended to other abiotic stresses to study the response of plant cells to the environment. PMID:27257817

  4. Enhanced osteoclastogenesis by mitochondrial retrograde signaling through transcriptional activation of the cathepsin K gene.

    PubMed

    Guha, Manti; Srinivasan, Satish; Koenigstein, Alexander; Zaidi, Mone; Avadhani, Narayan G

    2016-01-01

    Mitochondrial dysfunction has emerged as an important factor in wide ranging human pathologies. We have previously defined a retrograde signaling pathway that originates from dysfunctional mitochondria (Mt-RS) and causes a global nuclear transcriptional reprograming as its end point. Mitochondrial dysfunction causing disruption of mitochondrial membrane potential and consequent increase in cytosolic calcium [Ca(2) ](c) activates calcineurin and the transcription factors NF-κB, NFAT, CREB, and C/EBPδ. In macrophages, this signaling complements receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastic differentiation. Here, we show that the Mt-RS activated transcriptional coactivator heterogeneous ribonucleoprotein A2 (hnRNP A2) is induced by hypoxia in murine macrophages. We demonstrate that the cathepsin K gene (Ctsk), one of the key genes upregulated during osteoclast differentiation, is transcriptionally activated by Mt-RS factors. HnRNP A2 acts as a coactivator with nuclear transcription factors, cRel, and C/EBPδ for Ctsk promoter activation under hypoxic conditions. Notably, our study shows that hypoxia-induced activation of the stress target factors mediates effects similar to that of RANKL with regard to Ctsk activation. We therefore suggest that mitochondrial dysfunction and activation of Mt-RS, induced by various pathophysiologic conditions, is a potential risk factor for osteoclastogenesis and bone loss.

  5. Regulation of gene expression by manipulating transcriptional repressor activity using a novel CoSRI technology.

    PubMed

    Xu, Yue; Li, Song Feng; Parish, Roger W

    2016-12-20

    Targeted gene manipulation is a central strategy for studying gene function and identifying related biological processes. However, a methodology for manipulating the regulatory motifs of transcription factors is lacking as these factors commonly possess multiple motifs (eg. repression and activation motifs) which collaborate with each other to regulate multiple biological processes. We describe a novel approach designated Conserved Sequence-guided Repressor Inhibition (CoSRI) that can specifically reduce or abolish the repressive activities of transcription factors in vivo. The technology was evaluated using the chimeric MYB80-EAR transcription factor and subsequently the endogenous WUS transcription factor. The technology was employed to develop a reversible male sterility system applicable to hybrid seed production. In order to determine the capacity of the technology to regulate the activity of endogenous transcription factors, the WUS repressor was chosen. The WUS repression motif could be inhibited in vivo and the transformed plants exhibited the wus-1 phenotype. Consequently, the technology can be used to manipulate the activities of transcriptional repressor motifs regulating beneficial traits in crop plants and other eukaryotic organisms. This article is protected by copyright. All rights reserved.

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

    PubMed

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

    2017-01-01

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

  7. Synchronous activation of gonadotropin-releasing hormone gene transcription and secretion by pulsatile kisspeptin stimulation

    PubMed Central

    Choe, Han Kyoung; Kim, Hee-Dae; Park, Sung Ho; Lee, Han-Woong; Park, Jae-Yong; Seong, Jae Young; Lightman, Stafford L.; Son, Gi Hoon; Kim, Kyungjin

    2013-01-01

    Pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH) is essential for pituitary gonadotrope function. Although the importance of pulsatile GnRH secretion has been recognized for several decades, the mechanisms underlying GnRH pulse generation in hypothalamic neural networks remain elusive. Here, we demonstrate the ultradian rhythm of GnRH gene transcription in single GnRH neurons using cultured hypothalamic slices prepared from transgenic mice expressing a GnRH promoter-driven destabilized luciferase reporter. Although GnRH promoter activity in each GnRH neuron exhibited an ultradian pattern of oscillations with a period of ∼10 h, GnRH neuronal cultures exhibited partially synchronized bursts of GnRH transcriptional activity at ∼2-h intervals. Surprisingly, pulsatile administration of kisspeptin, a potent GnRH secretagogue, evoked dramatic synchronous activation of GnRH gene transcription with robust stimulation of pulsatile GnRH secretion. We also addressed the issue of hierarchical interaction between the circadian and ultradian rhythms by using Bmal1-deficient mice with defective circadian clocks. The circadian molecular oscillator barely affected basal ultradian oscillation of GnRH transcription but was heavily involved in kisspeptin-evoked responses of GnRH neurons. In conclusion, we have clearly shown synchronous bursts of GnRH gene transcription in the hypothalamic GnRH neuronal population in association with episodic neurohormone secretion, thereby providing insight into GnRH pulse generation. PMID:23509283

  8. TRIM33 switches off Ifnb1 gene transcription during the late phase of macrophage activation

    PubMed Central

    Ferri, Federica; Parcelier, Aude; Petit, Vanessa; Gallouet, Anne-Sophie; Lewandowski, Daniel; Dalloz, Marion; van den Heuvel, Anita; Kolovos, Petros; Soler, Eric; Squadrito, Mario Leonardo; De Palma, Michele; Davidson, Irwin; Rousselet, Germain; Romeo, Paul-Henri

    2015-01-01

    Despite its importance during viral or bacterial infections, transcriptional regulation of the interferon-β gene (Ifnb1) in activated macrophages is only partially understood. Here we report that TRIM33 deficiency results in high, sustained expression of Ifnb1 at late stages of toll-like receptor-mediated activation in macrophages but not in fibroblasts. In macrophages, TRIM33 is recruited by PU.1 to a conserved region, the Ifnb1 Control Element (ICE), located 15 kb upstream of the Ifnb1 transcription start site. ICE constitutively interacts with Ifnb1 through a TRIM33-independent chromatin loop. At late phases of lipopolysaccharide activation of macrophages, TRIM33 is bound to ICE, regulates Ifnb1 enhanceosome loading, controls Ifnb1 chromatin structure and represses Ifnb1 gene transcription by preventing recruitment of CBP/p300. These results characterize a previously unknown mechanism of macrophage-specific regulation of Ifnb1 transcription whereby TRIM33 is critical for Ifnb1 gene transcription shutdown. PMID:26592194

  9. TRIM33 switches off Ifnb1 gene transcription during the late phase of macrophage activation.

    PubMed

    Ferri, Federica; Parcelier, Aude; Petit, Vanessa; Gallouet, Anne-Sophie; Lewandowski, Daniel; Dalloz, Marion; van den Heuvel, Anita; Kolovos, Petros; Soler, Eric; Squadrito, Mario Leonardo; De Palma, Michele; Davidson, Irwin; Rousselet, Germain; Romeo, Paul-Henri

    2015-11-23

    Despite its importance during viral or bacterial infections, transcriptional regulation of the interferon-β gene (Ifnb1) in activated macrophages is only partially understood. Here we report that TRIM33 deficiency results in high, sustained expression of Ifnb1 at late stages of toll-like receptor-mediated activation in macrophages but not in fibroblasts. In macrophages, TRIM33 is recruited by PU.1 to a conserved region, the Ifnb1 Control Element (ICE), located 15 kb upstream of the Ifnb1 transcription start site. ICE constitutively interacts with Ifnb1 through a TRIM33-independent chromatin loop. At late phases of lipopolysaccharide activation of macrophages, TRIM33 is bound to ICE, regulates Ifnb1 enhanceosome loading, controls Ifnb1 chromatin structure and represses Ifnb1 gene transcription by preventing recruitment of CBP/p300. These results characterize a previously unknown mechanism of macrophage-specific regulation of Ifnb1 transcription whereby TRIM33 is critical for Ifnb1 gene transcription shutdown.

  10. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases.

    PubMed

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-03-01

    Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

  11. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

    PubMed Central

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-01-01

    Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals. PMID:26950874

  12. SUMOylation of DRIL1 Directs Its Transcriptional Activity Towards Leukocyte Lineage-Specific Genes

    PubMed Central

    van Lohuizen, Maarten; Peeper, Daniel S.

    2009-01-01

    DRIL1 is an ARID family transcription factor that can immortalize primary mouse fibroblasts, bypass RASV12-induced cellular senescence and collaborate with RASV12 or MYC in mediating oncogenic transformation. It also activates immunoglobulin heavy chain transcription and engages in heterodimer formation with E2F to stimulate E2F-dependent transcription. Little, however, is known about the regulation of DRIL1 activity. Recently, DRIL1 was found to interact with the SUMO-conjugating enzyme Ubc9, but the functional relevance of this association has not been assessed. Here, we show that DRIL1 is sumoylated both in vitro and in vivo at lysine 398. Moreover, we provide evidence that PIASy functions as a specific SUMO E3-ligase for DRIL1 and promotes its sumoylation both in vitro and in vivo. Furthermore, consistent with the subnuclear localization of PIASy in the Matrix-Associated Region (MAR), SUMO-modified DRIL1 species are found exclusively in the MAR fraction. This post-translational modification interferes neither with the subcellular localization nor the DNA-binding activity of the protein. In contrast, DRIL1 sumoylation impairs its interaction with E2F1 in vitro and modifies its transcriptional activity in vivo, driving transcription of subset of genes regulating leukocyte fate. Taken together, these results identify sumoylation as a novel post-translational modification of DRIL1 that represents an important mechanism for targeting and modulating DRIL1 transcriptional activity. PMID:19436740

  13. Rhodobacter sphaeroides LexA has dual activity: optimising and repressing recA gene transcription

    PubMed Central

    Tapias, Angels; Fernández, Silvia; Alonso, Juan C.; Barbé, Jordi

    2002-01-01

    Transcription of the Rhodobacter sphaeroides recA promoter (PrecA) is induced upon DNA damage in a lexA-dependent manner. In vivo experiments demonstrate that LexA protein represses and might also activate transcription of PrecA. Purified R.sphaeroides LexA protein specifically binds the SOS boxes located within the PrecA region. In vitro transcription analysis, using Escherichia coli RNA polymerase (RNAP), indicated that the presence of LexA may stimulate and repress transcription of PrecA. EMSA and DNase I footprinting experiments show that LexA and RNAP can bind simultaneously to PrecA. At low LexA concentrations it enhances RNAP binding to PrecA, stimulates open complex formation and strand separation beyond the transcription start site. At high LexA concentrations, however, RNAP-promoted strand separation is not observed beyond the +5 region. LexA might repress transcription by interfering with the clearance process instead of blocking the access of RNAP to the promoter region. Based on these findings we propose that the R.sphaeroides LexA protein performs fine tuning of the SOS response, which might provide a physiological advantage by enhancing transcription of SOS genes and delaying full activation of the response. PMID:11917014

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

    PubMed

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

    2009-02-17

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

  15. Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs).

    PubMed

    Lei, Yong; Guo, Xiaogang; Liu, Yun; Cao, Yang; Deng, Yi; Chen, Xiongfeng; Cheng, Christopher H K; Dawid, Igor B; Chen, Yonglong; Zhao, Hui

    2012-10-23

    Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F(1) frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.

  16. The product of the adenovirus intermediate gene IX is a transcriptional activator.

    PubMed Central

    Lutz, P; Rosa-Calatrava, M; Kedinger, C

    1997-01-01

    We have investigated the functional properties of the product of the adenovirus type 5 gene IX. This gene, which is expressed at intermediate times postinfection, encodes a small polypeptide (pIX) of 140 residues that has previously been shown to be incorporated into the viral capsid. Here, we show that pIX, in addition to its structural contribution, exhibits transcriptional properties. In transient transfection experiments, expression of pIX stimulated adenovirus major late promoter activity. The effect was independent of other viral proteins, but the level of promoter activation appeared strongly pIX dose dependent; similar levels of induction were observed with other cellular or viral TATA-containing (but not with TATA-less) promoters. This promoter specificity could be reproduced in a cell-free transcription system by the addition of purified recombinant pIX, further stressing the transcriptional nature of the phenomenon. A preliminary structural analysis of pIX indicated that the integrity of a putative leucine zipper at the carboxy-terminal end of the molecule, as well as elements within the amino-terminal half, was critical for pIX transcriptional activity. The relevance of these findings in adenovirus infection is discussed. PMID:9188576

  17. Autogenous transcriptional activation of a thiostrepton-induced gene in Streptomyces lividans.

    PubMed Central

    Holmes, D J; Caso, J L; Thompson, C J

    1993-01-01

    Although the antibiotic thiostrepton is best known as an inhibitor of protein synthesis, it also, at extremely low concentrations (< 10(-9) M), induces the expression of a regulon of unknown function in certain Streptomyces species. Here, we report the purification of a Streptomyces lividans thiostrepton-induced transcriptional activator protein, TipAL, whose N-terminus is similar to a family of eubacterial regulatory proteins represented by MerR. TipAL was first purified from induced cultures of S.lividans as a factor which bound to and activated transcription from its own promoter. The tipAL gene was overexpressed in Escherichia coli and TipAL protein purified in a single step using a thiostrepton affinity column. Thiostrepton enhanced binding of TipAL to the promoter and catalysed specific transcription in vitro. TipAS, a second gene product of the same open reading frame consisting of the C-terminal domain of TipAL, is apparently translated using its own in-frame initiation site. Since it is produced in large molar excess relative to TipAL after induction and also binds thiostrepton, it may competitively modulate transcriptional activation. Images PMID:7688297

  18. The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.

    PubMed

    Corbi, N; Libri, V; Fanciulli, M; Tinsley, J M; Davies, K E; Passananti, C

    2000-06-01

    Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

  19. Cloning and transcriptional activity analysis of the porcine cofilin 2 gene promoter.

    PubMed

    Wang, Jia-Mei; Lang, Bin; Zhu, Hong-yan; Du, Hai-ting; Tian, Yu-min; Su, Yu-hong

    2014-09-01

    Cofilins (CFL), including CFL1 and CFL2, are members of the family of actin-binding proteins in eukaryote. CFL2 is predominantly expressed in mammalian skeletal muscle and heart and is important to muscle fiber formation and muscular regeneration. To study transcriptional regulation of porcine CFL2, a 2.5 kb upstream sequence starting from the major CFL2 transcriptional start site was cloned by genome walking. Twelve DNA fragments of the 5' flank region of the porcine CFL2 gene were further isolated from porcine genomic DNA via PCR and inserted into the luciferase reporter vector pGL4.10 to make 12 CFL2 reporter constructs. All reporter vectors were transfected into C2C12, NIH3T3, or Hela cells and their relative luciferase activity measured after 48 h, respectively. Bioinformatics analysis suggested that there were two TATA-boxes at the -508 bp and -453 bp, as well as a GC-box and a CAAT-box in this sequence. Additional transcription factor binding sites including SP1, AP1, AP2, and GATA-1 sites were also predicted. The transcriptional activity of pGL4.10-1554 (1502 bp to +51 bp) was the strongest, and the promoter's active region was mapped to a region from -1502 bp to -1317 bp. Our data provide a foundation for future studies into transcriptional regulation of CFL2.

  20. PcaU, a Transcriptional Activator of Genes for Protocatechuate Utilization in Acinetobacter†

    PubMed Central

    Gerischer, Ulrike; Segura, Ana; Ornston, L. Nicholas

    1998-01-01

    The Acinetobacter pcaIJFBDKCHG operon encodes the six enzymes that convert protocatechuate to citric acid cycle intermediates. Directly downstream from the operon are qui and pob genes encoding sets of enzymes that convert quinate and p-hydroxybenzoate, respectively, to protocatechuate. Prior to this investigation, the only known regulatory gene in the pca-qui-pob cluster was pobR, which encodes a transcriptional activator that responds to p-hydroxybenzoate and activates transcription of pobA. The pca and qui genes were known to be expressed in response to protocatechuate, but a protein that mediated this induction had not been identified. This study was initiated by characterization of a spontaneous mutation that mapped upstream from pcaI and prevented expression of the pca genes. Sequencing of wild-type DNA extending from the translational start of pcaI through and beyond the location of the mutation revealed a 282-bp intergenic region and a divergently transcribed open reading frame, designated pcaU. Downstream from pcaU are two open reading frames encoding proteins similar in amino acid sequence to those associated with the oxidation of acyl thioesters. Inactivation of pcaU reduced the induced expression of pca structural genes by about 90% and impeded but did not completely prevent growth of the mutant cells with protocatechuate. PcaU was expressed in Escherichia coli and shown to bind to a portion of the pcaI-pcaU intergenic region containing a sequence identical in 16 of 19 nucleotide residues to a segment of the pob operator. Further similarity of the two regulatory systems is indicated by 54% amino acid sequence identity in the aligned primary structures of PobR and PcaU. The pob and pca systems were shown to differ, however, in the relative orientations of transcriptional starts with respect to the site where the activator binds to DNA, the size of the intergenic region, and the tightness of transcriptional control. The spontaneous mutation blocking pca

  1. Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation

    PubMed Central

    Weake, Vikki M.; Dyer, Jamie O.; Seidel, Christopher; Box, Andrew; Swanson, Selene K.; Peak, Allison; Florens, Laurence; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2011-01-01

    The Spt–Ada–Gcn5–acetyltransferase (SAGA) complex was discovered from Saccharomyces cerevisiae and has been well characterized as an important transcriptional coactivator that interacts both with sequence-specific transcription factors and the TATA-binding protein TBP. SAGA contains a histone acetyltransferase and a ubiquitin protease. In metazoans, SAGA is essential for development, yet little is known about the function of SAGA in differentiating tissue. We analyzed the composition, interacting proteins, and genomic distribution of SAGA in muscle and neuronal tissue of late stage Drosophila melanogaster embryos. The subunit composition of SAGA was the same in each tissue; however, SAGA was associated with considerably more transcription factors in muscle compared with neurons. Consistent with this finding, SAGA was found to occupy more genes specifically in muscle than in neurons. Strikingly, SAGA occupancy was not limited to enhancers and promoters but primarily colocalized with RNA polymerase II within transcribed sequences. SAGA binding peaks at the site of RNA polymerase pausing at the 5′ end of transcribed sequences. In addition, many tissue-specific SAGA-bound genes required its ubiquitin protease activity for full expression. These data indicate that in metazoans SAGA plays a prominent post-transcription initiation role in tissue-specific gene expression. PMID:21764853

  2. Transcriptional activation of the human cytotoxic serine protease gene CSP-B in T lymphocytes.

    PubMed Central

    Hanson, R D; Ley, T J

    1990-01-01

    The cytotoxic serine protease B (CSP-B) gene is activated during cytotoxic T-lymphocyte maturation. In this report, we demonstrate that the PEER T-cell line (bearing gamma/delta T-cell receptors) accumulates CSP-B mRNA following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) and N6-2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (bt2cAMP) because of transcriptional activation of the CSP-B gene. TPA and bt2cAMP act synergistically to induce CSP-B expression, since neither agent alone causes activation of CSP-B transcription or mRNA accumulation. Chromatin upstream from the CSP-B gene is resistant to DNase I digestion in untreated PEER cells, but becomes sensitive following TPA-bt2cAMP treatment. Upon activation of PEER cells, a DNase I-hypersensitive site forms upstream from the CSP-B gene within a region that is highly conserved in the mouse. Transient transfection of CSP-B promoter constructs identified two regulatory regions in the CSP-B 5'-flanking sequence, located at positions -609 to -202 and positions -202 to -80. The region from -615 to -63 is sufficient to activate a heterologous promoter in activated PEER cells, but activation is orientation specific, suggesting that this region behaves as an upstream promoter element rather than a classical enhancer. Consensus AP-1, AP-2, and cAMP response elements are found upstream from the CSP-B gene (as are several T-cell-specific consensus elements), but the roles of these elements in CSP-B gene activation have yet to be determined. Images PMID:2233710

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

    PubMed

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

    2005-02-01

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

  4. Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxin.

    PubMed

    Chandel, N S; Trzyna, W C; McClintock, D S; Schumacker, P T

    2000-07-15

    The transcription factor NF-kappa B stimulates the transcription of proinflammatory cytokines including TNF-alpha. LPS (endotoxin) and hypoxia both induce NF-kappa B activation and TNF-alpha gene transcription. Furthermore, hypoxia augments LPS induction of TNF-alpha mRNA. Previous reports have indicated that antioxidants abolish NF-kappa B activation in response to LPS or hypoxia, which suggests that reactive oxygen species (ROS) are involved in NF-kappa B activation. This study tested whether mitochondrial ROS are required for both NF-kappaB activation and the increase in TNF-alpha mRNA levels during hypoxia and LPS. Our results indicate that hypoxia (1.5% O2) stimulates NF-kappa B and TNF-alpha gene transcription and increases ROS generation as measured by the oxidant sensitive dye 2',7'-dichlorofluorescein diacetate in murine macrophage J774.1 cells. The antioxidants N-acetylcysteine and pyrrolidinedithiocarbamic acid abolished the hypoxic activation of NF-kappa B, TNF-alpha gene transcription, and increases in ROS levels. Rotenone, an inhibitor of mitochondrial complex I, abolished the increase in ROS signal, the activation of NF-kappa B, and TNF-alpha gene transcription during hypoxia. LPS stimulated NF-kappa B and TNF-alpha gene transcription but not ROS generation in J774.1 cells. Rotenone, pyrrolidinedithiocarbamic acid, and N-acetylcysteine had no effect on the LPS stimulation of NF-kappa B and TNF-alpha gene transcription, indicating that LPS activates NF-kappa B and TNF-alpha gene transcription through a ROS-independent mechanism. These results indicate that mitochondrial ROS are required for the hypoxic activation of NF-kappa B and TNF-alpha gene transcription, but not for the LPS activation of NF-kappa B.

  5. cis and trans activation of globin gene transcription in transient assays.

    PubMed

    Treisman, R; Green, M R; Maniatis, T

    1983-12-01

    We examined the effects of the simian virus 40 enhancer sequence on transcription of cloned human alpha- and beta-globin genes shortly after their introduction into cultured mammalian cells. We find that (i) detectable transcription of the beta-globin gene but not the alpha-globin gene requires linkage to the enhancer; (ii) the enhancer increases the amount of beta-globin RNA at least 100-fold but results in only a 5- to 10-fold increase in the amount of alpha-globin RNA; (iii) plasmid replication does not increase the level of beta-globin RNA, regardless of linkage to the enhancer, but does result in an approximately equal to 50-fold increase in the level of alpha-globin RNA; (iv) the enhancer is not required for and does not increase transcription of either gene in 293 cells, an adenovirus 5-transformed human kidney cell line. We also show that an enhancer sequence is not required for activity of the normally enhancer-dependent simian virus 40 early promoter in 293 cells, indicating that these cells contain a trans-acting factor(s) that circumvents the requirement for the enhancer sequence.

  6. Gene targeting in rats using transcription activator-like effector nucleases.

    PubMed

    Ménoret, Séverine; Tesson, Laurent; Rémy, Séverine; Usal, Claire; Thépenier, Virginie; Thinard, Reynald; Ouisse, Laure-Hélène; De Cian, Anne; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio

    2014-08-15

    The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

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

    PubMed

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

    2014-10-15

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

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

    PubMed Central

    Okada, Masahiro; Hirose, Susumu

    1998-01-01

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

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

    PubMed

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

    2016-09-15

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

  10. EGR1 regulates hepatic clock gene amplitude by activating Per1 transcription

    PubMed Central

    Tao, Weiwei; Wu, Jing; Zhang, Qian; Lai, Shan-Shan; Jiang, Shan; Jiang, Chen; Xu, Ying; Xue, Bin; Du, Jie; Li, Chao-Jun

    2015-01-01

    The mammalian clock system is composed of a master clock and peripheral clocks. At the molecular level, the rhythm-generating mechanism is controlled by a molecular clock composed of positive and negative feedback loops. However, the underlying mechanisms for molecular clock regulation that affect circadian clock function remain unclear. Here, we show that Egr1 (early growth response 1), an early growth response gene, is expressed in mouse liver in a circadian manner. Consistently, Egr1 is transactivated by the CLOCK/BMAL1 heterodimer through a conserved E-box response element. In hepatocytes, EGR1 regulates the transcription of several core clock genes, including Bmal1, Per1, Per2, Rev-erbα and Rev-erbβ, and the rhythm amplitude of their expression is dependent on EGR1’s transcriptional function. Further mechanistic studies indicated that EGR1 binds to the proximal region of the Per1 promoter to activate its transcription directly. When the peripheral clock is altered by light or feeding behavior transposition in Egr1-deficient mice, the expression phase of hepatic clock genes shifts normally, but the amplitude is also altered. Our data reveal a critical role for EGR1 in the regulation of hepatic clock circuitry, which may contribute to the rhythm stability of peripheral clock oscillators. PMID:26471974

  11. Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos.

    PubMed

    Lei, Yong; Guo, Xiaogang; Deng, Yi; Chen, Yonglong; Zhao, Hui

    2013-05-10

    Transcription activator-like effector nucleases (TALENs) are novel engineered DNA nucleases, and have been proven to be effective for gene specific targeting in various species. Recently we reported gene disruptions in Xenopus embryos by using TALENs. Here we summarize the protocol that is used in our studies for gene disruption. This protocol covers selection of TALEN targeting sites, TALEN assembly with a modified Golden Gate method, and injection of TALEN mRNAs into Xenopus tropicalis embryos. We also provide details for detection of somatic and germ line transmitted mutations. And finally, we briefly describe establishment of knockout Xenopus lines. This protocol will facilitate broader applications of TALENs in studies of Xenopus biology.

  12. Assessment of Transcriptional Activity of Borrelia burgdorferi and Host Cytokine Genes During Early and Late Infection in a Mouse Model

    PubMed Central

    Feng, Sunlian; Barthold, Stephen W.

    2013-01-01

    Abstract Differential gene expression by Borrelia burgdorferi spirochetes during mammalian infection facilitates their dissemination as well as immune evasion. Modulation of gene transcription in response to host immunity has been documented with the outer surface protein C, but the influence of transcription of other genes is largely unknown. A low-density array (LDA) was developed to study transcriptional activity of 43 B. burgdorferi genes and 19 host genes that may be involved in various host–agent interactions. Gene transcription in heart, joint, and muscle tissue was compared in immunocompetent C3H and immunodeficient C3H-scid mice during early (3 weeks) and late (2 months) B. burgdorferi infection. Among all tissue types, levels of relative transcription of over 80% of B. burgdorferi genes tested were one- to nine-fold less in C3H mice compared to C3H-scid mice. At the later time point, all genes were transcribed in C3H-scid mice, whereas transcription of 16 genes out of 43 tested was not detected in analyzed tissues of C3H mice. Our data suggest that during infection of immunocompetent mice, a majority of B. burgdorferi genes tested are downregulated in response to acquired host immunity. LDA revealed variable patterns of host gene expression in different tissues and at different intervals in infected mice. Higher levels of relative expression for IL-10 during both early and late infection were detected in heart base, and it was unchanged in the tibiotarsal joint. Comparative analysis of B. burgdorferi and host genes transcriptional activity revealed that increased flaB mRNA during early infection was followed by increases of CCL7, CCL8, interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) in all assessed tissue types. LDA represents a valuable approach for sensitive and quantitative gene transcription profiling and for understanding Lyme borreliosis. PMID:23930938

  13. The cancer gene WWOX behaves as an inhibitor of SMAD3 transcriptional activity via direct binding

    PubMed Central

    2013-01-01

    Background The WW domain containing protein WWOX has been postulated to behave as a tumor suppressor in breast and other cancers. Expression of this protein is lost in over 70% of ER negative tumors. This prompted us to investigate the phenotypic and gene expression effects of loss of WWOX expression in breast cells. Methods Gene expression microarrays and standard in vitro assays were performed on stably silenced WWOX (shRNA) normal breast cells. Bioinformatic analyses were used to identify gene networks and transcriptional regulators affected by WWOX silencing. Co-immunoprecipitations and GST-pulldowns were used to demonstrate a direct interaction between WWOX and SMAD3. Reporter assays, ChIP, confocal microscopy and in silico analyses were employed to determine the effect of WWOX silencing on TGFβ-signaling. Results WWOX silencing affected cell proliferation, motility, attachment and deregulated expression of genes involved in cell cycle, motility and DNA damage. Interestingly, we detected an enrichment of targets activated by the SMAD3 transcription factor, including significant upregulation of ANGPTL4, FST, PTHLH and SERPINE1 transcripts. Importantly, we demonstrate that the WWOX protein physically interacts with SMAD3 via WW domain 1. Furthermore, WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promoters and significantly quenches activation of a TGFβ responsive reporter. Additionally, WWOX expression leads to redistribution of SMAD3 from the nuclear to the cytoplasmic compartment. Since the TGFβ target ANGPTL4 plays a key role in lung metastasis development, we performed a meta-analysis of ANGPTL4 expression relative to WWOX in microarray datasets from breast carcinomas. We observed a significant inverse correlation between WWOX and ANGPTL4. Furthermore, the WWOX lo /ANGPTL4 hi cluster of breast tumors is enriched in triple-negative and basal-like sub-types. Tumors with this gene expression signature could represent

  14. Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

    PubMed

    Gersbach, Charles A; Perez-Pinera, Pablo

    2014-08-01

    New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

  15. Editing of the heavy chain gene of Bombyx mori using transcription activator like effector nucleases.

    PubMed

    Wang, Yujun; Nakagaki, Masao

    2014-07-18

    The silk gland of Bombyx mori represents an established in vivo system for producing recombinant proteins. However, low yields of recombinant proteins have limited the system's further development because endogenous silk proteins were present. Transcription activator-like effector nucleases (TALENs) tool which work in pairs to bind and cleave DNA at specific sites, have recently been shown to be effective for genome editing in various organisms, including silkworms. To improve the yield of recombinant proteins synthesized in the silkworm by eliminated competition with endogenous fibroin synthesis, the heavy chain (H-chain) gene was knocked out using transcription activator-like effector nucleases (TALENs). A pair of TALENs that targets the 1st exon in the H-chain gene was synthesized and microinjected into silkworm embryos; the injected silkworms were screened for H-chain gene knock out (H-KO) based on their sericin cocoon-making characteristics. Sequence analysis revealed that the H-chain of the mutation was successfully edited. The TALENs was very efficient in editing the genome DNA of silkworm. By being eliminated competition with the H-chain, the production of recombinant proteins would be expected to increase markedly if this H-KO system is used.

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

    PubMed Central

    Berchtold, Evi; Csaba, Gergely; Zimmer, Ralf

    2016-01-01

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

  17. DNase I sensitivity of transcriptionally active genes in intact nuclei and isolated chromatin of plants.

    PubMed Central

    Spiker, S; Murray, M G; Thompson, W F

    1983-01-01

    We have investigated the DNase I sensitivity of transcriptionally active DNA sequences in intact nuclei and isolated chromatin from embryos of wheat (Triticum aestivum L.). Nuclei or isolated chromatin was incubated with DNase I, and the extent of DNA digestion was monitored as percentage acid solubility. The resistant DNA and DNA from sham-digested controls were used to drive reassociation reactions with cDNA populations corresponding to either total poly(A)+RNA from unimbibed wheat embryos or polysomal poly(A)+RNA from embryos that had imbibed for 3 hr. Sequences complementary to either probe were depleted in DNase I-resistant DNA from nuclei and from chromatin isolated under low-ionic-strength conditions. This indicates that transcriptionally active sequences are preferentially DNase I sensitive in plants. In chromatin isolated at higher ionic strength, cDNA complementary sequences were not preferentially depleted by DNase I treatment. Therefore, the chromatin structure that confers preferential DNase I sensitivity to transcriptionally active genes appears to be lost when the higher-ionic-strength method of preparation is used. Treatment of wheat nuclei with DNase I causes the release of four prominent nonhistone chromosomal proteins that comigrate with wheat high mobility group proteins on NaDodSO4 gels. Images PMID:6219388

  18. Shaping synaptic plasticity: the role of activity-mediated epigenetic regulation on gene transcription.

    PubMed

    Cortés-Mendoza, Javier; Díaz de León-Guerrero, Sol; Pedraza-Alva, Gustavo; Pérez-Martínez, Leonor

    2013-10-01

    Learning and memory are basic functions of the brain that allowed human evolution. It is well accepted that during learning and memory formation the dynamic establishment of new active synaptic connections is crucial. Persistent synaptic activation leads to molecular events that include increased release of neurotransmitters, increased expression of receptors on the postsynaptic neuron, thus creating a positive feedback that results in the activation of distinct signaling pathways that temporally and permanently alter specific patterns of gene expression. However, the epigenetic changes that allow the establishment of long term genetic programs that control learning and memory are not completely understood. Even less is known regarding the signaling events triggered by synaptic activity that regulate these epigenetic marks. Here we review the current understanding of the molecular mechanisms controlling activity-dependent gene transcription leading synaptic plasticity and memory formation. We describe how Ca(2+) entry through N-methyl-d-aspartate-type glutamate neurotransmitter receptors result in the activation of specific signaling pathways leading to changes in gene expression, giving special emphasis to the recent data pointing out different epigenetic mechanisms (histone acetylation, methylation and phosphorylation as well as DNA methylation and hydroxymethylation) underlying learning and memory.

  19. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases.

    PubMed

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-06-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

  20. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases

    PubMed Central

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background. PMID:23630316

  1. Transcriptional activation of Xenopus class III genes in chromatin isolated from sperm and somatic nuclei.

    PubMed Central

    Wolffe, A P

    1989-01-01

    Xenopus sperm chromatin lacks class III transcription complexes and somatic histone H1. Inactive class III genes in sperm chromatin are easily programmed with transcription complexes de novo and transcribed in Xenopus oocyte nuclear extract. In contrast, repressed class III genes in somatic chromatin are not transcribed in the oocyte nuclear extract. Class III genes that are initially inactive or repressed in both types of chromatin can be efficiently transcribed in a cell free preparation of Xenopus eggs. Chromatin mediated repression of class III genes in somatic nuclei is reversible in Xenopus egg extract, but not in the oocyte nuclear extract. Any inhibition of transcription attributed to chromatin assembly onto a gene, will therefore depend on the extract in which transcription is assayed. Images PMID:2915929

  2. Activation of gene transcription via CIM0216, a synthetic ligand of transient receptor potential melastatin-3 (TRPM3) channels.

    PubMed

    Rubil, Sandra; Thiel, Gerald

    2017-01-02

    Several compounds have been proposed to stimulate TRPM3 Ca(2+) channels. We recently showed that stimulation of TRPM3 channels with pregnenolone sulfate activated the transcription factor AP-1, while other proposed TRPM3 ligands (nifedipine, D-erythro-sphingosine) exhibited either no or TRPM3-independent effects on gene transcription. Here, we have analyzed the transcriptional activity of CIM0216, a synthetic TRPM3 ligand proposed to have a higher potency and affinity for TRPM3 than pregnenolone sulfate. The results show that CIM0216 treatment of HEK293 cells expressing TRPM3 channels activated AP-1 and stimulated the transcriptional activation potential of c-Jun and c-Fos, 2 basic region leucine zipper transcription factors that constitute AP-1. CIM0216-induced gene transcription was attenuated by knock-down of TRPM3 or treatment with mefenamic acid, a TRPM3 inhibitor. CIM0216 was similarly or less capable in activating TRPM3-mediated gene transcription, suggesting that pregnenolone sulfate is still the ligand of choice for changing the gene expression pattern via TRPM3.

  3. Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila.

    PubMed

    Beumer, Kelly J; Trautman, Jonathan K; Christian, Michelle; Dahlem, Timothy J; Lake, Cathleen M; Hawley, R Scott; Grunwald, David J; Voytas, Daniel F; Carroll, Dana

    2013-10-03

    Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.

  4. Transcript degradation and noise of small RNA-controlled genes in a switch activated network in Escherichia coli.

    PubMed

    Arbel-Goren, Rinat; Tal, Asaf; Parasar, Bibudha; Dym, Alvah; Costantino, Nina; Muñoz-García, Javier; Court, Donald L; Stavans, Joel

    2016-08-19

    Post-transcriptional regulatory processes may change transcript levels and affect cell-to-cell variability or noise. We study small-RNA downregulation to elucidate its effects on noise in the iron homeostasis network of Escherichia coli In this network, the small-RNA RyhB undergoes stoichiometric degradation with the transcripts of target genes in response to iron stress. Using single-molecule fluorescence in situ hybridization, we measured transcript numbers of the RyhB-regulated genes sodB and fumA in individual cells as a function of iron deprivation. We observed a monotonic increase of noise with iron stress but no evidence of theoretically predicted, enhanced stoichiometric fluctuations in transcript numbers, nor of bistable behavior in transcript distributions. Direct detection of RyhB in individual cells shows that its noise is much smaller than that of these two targets, when RyhB production is significant. A generalized two-state model of bursty transcription that neglects RyhB fluctuations describes quantitatively the dependence of noise and transcript distributions on iron deprivation, enabling extraction of in vivo RyhB-mediated transcript degradation rates. The transcripts' threshold-linear behavior indicates that the effective in vivo interaction strength between RyhB and its two target transcripts is comparable. Strikingly, the bacterial cell response exhibits Fur-dependent, switch-like activation instead of a graded response to iron deprivation.

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

    PubMed

    Bhat, R V; Baraban, J M

    1993-10-01

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

  6. Transcriptional Regulation of the SMK1 Mitogen-Activated Protein Kinase Gene during Meiotic Development in Saccharomyces cerevisiae

    PubMed Central

    Pierce, Michael; Wagner, Marisa; Xie, Jianxin; Gailus-Durner, Valérie; Six, John; Vershon, Andrew K.; Winter, Edward

    1998-01-01

    Meiotic development (sporulation) in Saccharomyces cerevisiae is characterized by an ordered pattern of gene expression, with sporulation-specific genes classified as early, middle, mid-late, or late depending on when they are expressed. SMK1 encodes a mitogen-activated protein kinase required for spore morphogenesis that is expressed as a middle sporulation-specific gene. Here, we identify the cis-acting DNA elements that regulate SMK1 transcription and characterize the phenotypes of mutants with altered expression patterns. The SMK1 promoter contains an upstream activating sequence (UASS) that specifically interacts with the transcriptional activator Abf1p. The Abf1p-binding sites from the early HOP1 and the middle SMK1 promoters are functionally interchangeable, demonstrating that these elements do not play a direct role in their differential transcriptional timing. Timing of SMK1 expression is determined by another cis-acting DNA sequence termed MSE (for middle sporulation element). The MSE is required not only for activation of SMK1 transcription during middle sporulation but also for its repression during vegetative growth and early meiosis. In addition, the SMK1 MSE can repress vegetative expression in the context of the HOP1 promoter and convert HOP1 from an early to a middle gene. SMK1 function is not contingent on its tight transcriptional regulation as a middle sporulation-specific gene. However, promoter mutants with different quantitative defects in SMK1 transcript levels during middle sporulation show distinct sporulation phenotypes. PMID:9742114

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

    PubMed

    Yan, C; Whitsett, J A

    1997-07-11

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

  8. Rcs signalling-activated transcription of rcsA induces strong anti-sense transcription of upstream fliPQR flagellar genes from a weak intergenic promoter: regulatory roles for the anti-sense transcript in virulence and motility.

    PubMed

    Wang, Qingfeng; Harshey, Rasika M

    2009-10-01

    In Salmonella enterica, an activated Rcs signalling system inhibits initiation of transcription of the flhD master operon. Under these conditions, where motility is shut down, microarray experiments showed an increased RNA signal for three flagellar genes -fliPQR- located upstream of rcsA. We show here that it is the anti-sense (AS) strand of these genes that is transcribed, originating at a weak promoter in the intergenic region between fliR and rcsA. RcsA is an auxiliary regulator for the Rcs system, whose transcription is dependent on the response regulator RcsB. Rcs-activated rightward transcription, but not translation, of rcsA is required for stimulation of leftward AS transcription. Our results implicate a combined action of RcsB and rcsA transcription in activating the AS promoter, likely by modulating DNA superhelicity in the intergenic region. We show that the AS transcript regulates many genes in the Rcs regulon, including SPI-1 and SPI-2 virulence and stress-response genes. In the wild-type strain the AS transcript is present in low amounts, independent of Rcs signalling. Here, AS transcription modulates complementary sense RNA levels and impacts swarming motility. It appears that the flagellar AS transcript has been co-opted by the Rcs system to regulate virulence.

  9. Transcriptional activation by heat and cold of a thiol protease gene in tomato. [Lycopersicon esculentum

    SciTech Connect

    Schaffer, M.A.; Fischer, R.L. )

    1990-08-01

    We previously determined that low temperature induces the accumulation in tomato (Lycopersicon esculentum) fruit of a cloned mRNA, designated C14, encoding a polypeptide related to thiol proteases. We now demonstrate that C14 mRNA accumulation is a response common to both high (40{degree}C) and low (4{degree}C) temperature stresses. Exposure of tomato fruit to 40{degree}C results in the accumulation of C14 mRNA, by 8 hours. This response is more rapid than that to 4{degree}C, but slower than the induction of many heat shock messages by 40{degree}C, and therefore unique. We have also studied the mechanism by which heat and cold exposure activate C14 gene expression. Both high and low temperature regulate protease gene expression through transcriptional induction of a single C14 gene. A hypothesis for the function of C14 thiol protease gene expression in response to heat and cold is discussed.

  10. Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

    PubMed Central

    Almeida, Luis E. F.; Murray, Peter D.; Zielke, H. Ronald; Roby, Clinton D.; Kingsbury, Tami J.; Krueger, Bruce K.

    2009-01-01

    Cyclic AMP can stimulate the transcription of many activity-dependent genes via activation of the transcription factor, CREB. However, in mouse cortical neuron cultures, prior to synaptogenesis, neither cAMP nor dopamine, which acts via cAMP, stimulated CREB-dependent gene transcription when NR2B-containing NMDA receptors (NMDARs) were blocked. Stimulation of transcription by cAMP was potentiated by inhibitors of excitatory amino acid uptake, suggesting a role for extracellular glutamate or aspartate in cAMP-induced transcription. Aspartate was identified as the extracellular messenger: enzymatic scavenging of L-aspartate, but not glutamate, blocked stimulation of CREB-dependent gene transcription by cAMP; moreover, cAMP induced aspartate but not glutamate release. Taken together, these results suggest that cAMP acts via an autocrine or paracrine pathway to release aspartate, which activates NR2B-containing NMDARs, leading to Ca2+ entry and activation of transcription. This cAMP/aspartate/NMDAR signaling pathway may mediate the effects of transmitters such as dopamine on axon growth and synaptogenesis in developing neurons or on synaptic plasticity in mature neural networks. PMID:19812345

  11. Binding of TFIIIC to SINE Elements Controls the Relocation of Activity-Dependent Neuronal Genes to Transcription Factories

    PubMed Central

    Crepaldi, Luca; Policarpi, Cristina; Coatti, Alessandro; Sherlock, William T.; Jongbloets, Bart C.; Down, Thomas A.; Riccio, Antonella

    2013-01-01

    In neurons, the timely and accurate expression of genes in response to synaptic activity relies on the interplay between epigenetic modifications of histones, recruitment of regulatory proteins to chromatin and changes to nuclear structure. To identify genes and regulatory elements responsive to synaptic activation in vivo, we performed a genome-wide ChIPseq analysis of acetylated histone H3 using somatosensory cortex of mice exposed to novel enriched environmental (NEE) conditions. We discovered that Short Interspersed Elements (SINEs) located distal to promoters of activity-dependent genes became acetylated following exposure to NEE and were bound by the general transcription factor TFIIIC. Importantly, under depolarizing conditions, inducible genes relocated to transcription factories (TFs), and this event was controlled by TFIIIC. Silencing of the TFIIIC subunit Gtf3c5 in non-stimulated neurons induced uncontrolled relocation to TFs and transcription of activity-dependent genes. Remarkably, in cortical neurons, silencing of Gtf3c5 mimicked the effects of chronic depolarization, inducing a dramatic increase of both dendritic length and branching. These findings reveal a novel and essential regulatory function of both SINEs and TFIIIC in mediating gene relocation and transcription. They also suggest that TFIIIC may regulate the rearrangement of nuclear architecture, allowing the coordinated expression of activity-dependent neuronal genes. PMID:23966877

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

    SciTech Connect

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

    2008-08-01

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

  13. Transcript degradation and noise of small RNA-controlled genes in a switch activated network in Escherichia coli

    PubMed Central

    Arbel-Goren, Rinat; Tal, Asaf; Parasar, Bibudha; Dym, Alvah; Costantino, Nina; Muñoz-García, Javier; Court, Donald L.; Stavans, Joel

    2016-01-01

    Post-transcriptional regulatory processes may change transcript levels and affect cell-to-cell variability or noise. We study small-RNA downregulation to elucidate its effects on noise in the iron homeostasis network of Escherichia coli. In this network, the small-RNA RyhB undergoes stoichiometric degradation with the transcripts of target genes in response to iron stress. Using single-molecule fluorescence in situ hybridization, we measured transcript numbers of the RyhB-regulated genes sodB and fumA in individual cells as a function of iron deprivation. We observed a monotonic increase of noise with iron stress but no evidence of theoretically predicted, enhanced stoichiometric fluctuations in transcript numbers, nor of bistable behavior in transcript distributions. Direct detection of RyhB in individual cells shows that its noise is much smaller than that of these two targets, when RyhB production is significant. A generalized two-state model of bursty transcription that neglects RyhB fluctuations describes quantitatively the dependence of noise and transcript distributions on iron deprivation, enabling extraction of in vivo RyhB-mediated transcript degradation rates. The transcripts’ threshold-linear behavior indicates that the effective in vivo interaction strength between RyhB and its two target transcripts is comparable. Strikingly, the bacterial cell response exhibits Fur-dependent, switch-like activation instead of a graded response to iron deprivation. PMID:27085802

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

    PubMed Central

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

    2014-01-01

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

  15. GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes.

    PubMed

    Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

    2014-09-01

    Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.

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

    PubMed

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

    2017-01-29

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

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

    PubMed

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

    2016-08-01

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

  18. Transcriptional control of muscle plasticity: differential regulation of troponin I genes by electrical activity.

    PubMed

    Calvo, S; Stauffer, J; Nakayama, M; Buonanno, A

    1996-01-01

    Plasticity of the skeletal muscle phenotype can result from the selective repression and activation of gene expression in response to innervation patterns. Motoneurons, eliciting different patterns of depolarization, regulate the contractile properties of the myofibers they innervate by selectively activating expression of genes encoding fiber-type-specific (fast vs. slow) contractile proteins. We have analyzed the regulation of the troponin I slow (TnIs) and fast (TnIf) genes as a model to study the molecular mechanisms regulating fiber-type plasticity. We found that expression of the two TnI isoforms is downregulated by denervation. Moreover, TnI expression is upregulated by specific patterns of electrical activity [10 Hz vs. 100 Hz] used to depolarize muscle. We previously isolated the rat TnIs gene and demonstrated that regulatory sequences reside in its upstream region and second intron [Banerjee-Basu S, Buonanno A (1993), Mol Cell Biol 12:5024-5032]. Using transgenic mice, we show that the upstream region of the TnIs gene extending from -949 to +50 is sufficient to confer transcription specifically in slowtwitch muscles. Serial deletions of the TnIs upstream and intronic regions were generated in a CAT reporter vector to delineate transcriptional regulatory elements in transiently transfected Sol8 myotubes. Sequences necessary to confer the highest levels of TnIs transcription mapped to the upstream region between -0.95 and -0.72 kb, and to a 56 bp sequence located in the second intron. Comparison of the at sequence between -0.95 and -0.72 to the human TnIs gene identified a highly homologous region of 128 bp that we named the TnI SURE (slow upstream regulatory element). Alignment of these two SURE sequences with the quail TnI fast intronic regulatory element identified common motifs, namely, two A/T-rich sequences (A/T1 and A/T2) with homology to homeotic protein and MEF2 binding sites, a CACC box, an E box, and a novel motif (GCAGGCA) that we denoted the

  19. Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations

    PubMed Central

    Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

    2017-01-01

    Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators. PMID:28301511

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

    PubMed

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

    2015-09-11

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

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

    PubMed Central

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

    1988-01-01

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

  2. Transcription initiation in vivo without classical transactivators: DNA kinks flanking the core promoter of the housekeeping yeast adenylate kinase gene, AKY2, position nucleosomes and constitutively activate transcription.

    PubMed

    Angermayr, Michaela; Oechsner, Ulrich; Gregor, Kerstin; Schroth, Gary P; Bandlow, Wolfhard

    2002-10-01

    The housekeeping gene of the major adenylate kinase in Saccharomyces cerevisiae (AKY2, ADK1) is constitutively transcribed at a moderate level. The promoter has been dissected in order to define elements that effect constitutive transcription. Initiation of mRNA synthesis at the AKY2 promoter is shown to be mediated by a non-canonic core promoter, (TA)(6). Nucleotide sequences 5' of this element only marginally affect transcription suggesting that promoter activation can dispense with transactivators and essentially involves basal transcription. We show that the core promoter of AKY2 is constitutively kept free of nucleosomes. Analyses of permutated AKY2 promoter DNA revealed the presence of bent DNA. DNA structure analysis by computer and by mutation identified two kinks flanking an interstitial stretch of 65 bp of moderately bent core promoter DNA. Kinked DNA is likely incompatible with packaging into nucleosomes and responsible for positioning nucleosomes at the flanks allowing unimpeded access of the basal transcription machinery to the core promoter. The data show that in yeast, constitutive gene expression can dispense with classical transcriptional activator proteins, if two prerequisites are met: (i) the core promoter is kept free of nucleosomes; this can be due to structural properties of the DNA as an alternative to chromatin remodeling factors; and (ii) the core promoter is pre-bent to allow a high rate of basal transcription initiation.

  3. Rapid and efficient assembly of transcription activator-like effector genes by USER cloning.

    PubMed

    Wang, Song; Li, Wei; Wang, Shuo; Hu, Baoyang

    2014-06-20

    Transcription activator-like effectors (TALEs) that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent (USER), an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases (TALENs) generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tet1 locus, and proved that all can specifically target Tet1 gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.

  4. Analysis of gene transcription in cells lacking DNA-PK activity.

    PubMed

    Bryntesson, F; Regan, J C; Jeggo, P A; Taccioli, G E; Hubank, M

    2001-08-01

    The DNA-dependent protein kinase (DNA-PK), comprised of the Ku70/Ku80 (now known as G22p1/Xrcc5) heterodimer and the catalytic subunit DNA-PKcs (now known as Prkdc), is required for the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair. The mechanism of action of DNA-PK remains unclear. We have investigated whether DNA-PK regulates gene transcription in vivo after DNA damage using the subtractive hybridization technique of cDNA representational difference analysis (cDNA RDA). Differential transcription, both radiation-dependent and independent, was detected and confirmed in primary mouse embryo fibroblasts from DNA-PKcs(-/-) and DNA-PKcs(+/+) mice. We present evidence that transcription of the extracellular matrix gene laminin alpha 4 (Lama4) is regulated by DNA-PK in a radiation-independent manner. However, screening of both primary and immortalized DNA-PKcs-deficient cell lines demonstrates that the majority of differences were not consistently dependent on DNA-PK status. Similar results were obtained in experiments using KU mutant hamster cell lines, indicating heterogeneity of transcription between closely related cell lines. Our results suggest that while DNA-PK may be involved in limited gene-specific transcription, it does not play a major role in the transcriptional response to DNA damage.

  5. Nuclear localization domains of GATA activator Gln3 are required for transcription of target genes through dephosphorylation in Saccharomyces cerevisiae.

    PubMed

    Numamoto, Minori; Tagami, Shota; Ueda, Yusuke; Imabeppu, Yusuke; Sasano, Yu; Sugiyama, Minetaka; Maekawa, Hiromi; Harashima, Satoshi

    2015-08-01

    The GATA transcription activator Gln3 in the budding yeast (Saccharomyces cerevisiae) activates transcription of nitrogen catabolite repression (NCR)-sensitive genes. In cells grown in the presence of preferred nitrogen sources, Gln3 is phosphorylated in a TOR-dependent manner and localizes in the cytoplasm. In cells grown in non-preferred nitrogen medium or treated with rapamycin, Gln3 is dephosphorylated and is transported from the cytoplasm to the nucleus, thereby activating the transcription of NCR-sensitive genes. Caffeine treatment also induces dephosphorylation of Gln3 and its translocation to the nucleus and transcription of NCR-sensitive genes. However, the details of the mechanism by which phosphorylation controls Gln3 localization and transcriptional activity are unknown. Here, we focused on two regions of Gln3 with nuclear localization signal properties (NLS-K, and NLS-C) and one with nuclear export signal (NES). We constructed various mutants for our analyses: gln3 containing point mutations in all potential phosphoacceptor sites (Thr-339, Ser-344, Ser-347, Ser-355, Ser-391) in the NLS and NES regions to produce non-phosphorylatable (alanine) or mimic-phosphorylatable (aspartic acid) residues; and deletion mutants. We found that phosphorylation of Gln3 was impaired in all of these mutations and that the aspartic acid substitution mutants showed drastic reduction of Gln3-mediated transcriptional activity despite the fact that the mutations had no effect on nuclear localization of Gln3. Our observations suggest that these regions are required for transcription of target genes presumably through dephosphorylation.

  6. Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

    PubMed Central

    Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

    2016-01-01

    Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage. PMID:26613339

  7. Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages.

    PubMed

    Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

    2016-05-01

    Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage.

  8. Crystal structure of the caseinolytic protease gene regulator, a transcriptional activator in actinomycetes.

    PubMed

    Russo, Santina; Schweitzer, Jens-Eric; Polen, Tino; Bott, Michael; Pohl, Ehmke

    2009-02-20

    Human pathogens of the genera Corynebacterium and Mycobacterium possess the transcriptional activator ClgR (clp gene regulator) which in Corynebacterium glutamicum has been shown to regulate the expression of the ClpCP protease genes. ClgR specifically binds to pseudo-palindromic operator regions upstream of clpC and clpP1P2. Here, we present the first crystal structure of a ClgR protein from C. glutamicum. The structure was determined from two different crystal forms to resolutions of 1.75 and 2.05 A, respectively. ClgR folds into a five-helix bundle with a helix-turn-helix motif typical for DNA-binding proteins. Upon dimerization the two DNA-recognition helices are arranged opposite to each other at the protein surface in a distance of approximately 30 A, which suggests that they bind into two adjacent major grooves of B-DNA in an anti-parallel manner. A binding pocket is situated at a strategic position in the dimer interface and could possess a regulatory role altering the positions of the DNA-binding helices.

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

    PubMed

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

    2014-04-01

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

  10. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).

    PubMed

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F; Hur, Man-Wook

    2008-10-24

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation.

  11. Transcriptional Activation of Low-Density Lipoprotein Receptor Gene by DJ-1 and Effect of DJ-1 on Cholesterol Homeostasis

    PubMed Central

    Takahashi-Niki, Kazuko; Kato, Izumi; Niki, Takeshi; Goldberg, Matthew S.; Shen, Jie; Ishimoto, Kenji; Doi, Takefumi; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2012-01-01

    DJ-1 is a novel oncogene and also causative gene for familial Parkinson’s disease park7. DJ-1 has multiple functions that include transcriptional regulation, anti-oxidative reaction and chaperone and mitochondrial regulation. For transcriptional regulation, DJ-1 acts as a coactivator that binds to various transcription factors, resulting in stimulation or repression of the expression of their target genes. In this study, we found the low-density lipoprotein receptor (LDLR) gene is a transcriptional target gene for DJ-1. Reduced expression of LDLR mRNA and protein was observed in DJ-1-knockdown cells and DJ-1-knockout mice and this occurred at the transcription level. Reporter gene assays using various deletion and point mutations of the LDLR promoter showed that DJ-1 stimulated promoter activity by binding to the sterol regulatory element (SRE) with sterol regulatory element binding protein (SREBP) and that stimulating activity of DJ-1 toward LDLR promoter activity was enhanced by oxidation of DJ-1. Chromatin immunoprecipitation, gel-mobility shift and co-immunoprecipitation assays showed that DJ-1 made a complex with SREBP on the SRE. Furthermore, it was found that serum LDL cholesterol level was increased in DJ-1-knockout male, but not female, mice and that the increased serum LDL cholesterol level in DJ-1-knockout male mice was cancelled by administration with estrogen, suggesting that estrogen compensates the increased level of serum LDL cholesterol in DJ-1-knockout female mice. This is the first report that DJ-1 participates in metabolism of fatty acid synthesis through transcriptional regulation of the LDLR gene. PMID:22666465

  12. Transcriptional activation of low-density lipoprotein receptor gene by DJ-1 and effect of DJ-1 on cholesterol homeostasis.

    PubMed

    Yamaguchi, Shiori; Yamane, Takuya; Takahashi-Niki, Kazuko; Kato, Izumi; Niki, Takeshi; Goldberg, Matthew S; Shen, Jie; Ishimoto, Kenji; Doi, Takefumi; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2012-01-01

    DJ-1 is a novel oncogene and also causative gene for familial Parkinson's disease park7. DJ-1 has multiple functions that include transcriptional regulation, anti-oxidative reaction and chaperone and mitochondrial regulation. For transcriptional regulation, DJ-1 acts as a coactivator that binds to various transcription factors, resulting in stimulation or repression of the expression of their target genes. In this study, we found the low-density lipoprotein receptor (LDLR) gene is a transcriptional target gene for DJ-1. Reduced expression of LDLR mRNA and protein was observed in DJ-1-knockdown cells and DJ-1-knockout mice and this occurred at the transcription level. Reporter gene assays using various deletion and point mutations of the LDLR promoter showed that DJ-1 stimulated promoter activity by binding to the sterol regulatory element (SRE) with sterol regulatory element binding protein (SREBP) and that stimulating activity of DJ-1 toward LDLR promoter activity was enhanced by oxidation of DJ-1. Chromatin immunoprecipitation, gel-mobility shift and co-immunoprecipitation assays showed that DJ-1 made a complex with SREBP on the SRE. Furthermore, it was found that serum LDL cholesterol level was increased in DJ-1-knockout male, but not female, mice and that the increased serum LDL cholesterol level in DJ-1-knockout male mice was cancelled by administration with estrogen, suggesting that estrogen compensates the increased level of serum LDL cholesterol in DJ-1-knockout female mice. This is the first report that DJ-1 participates in metabolism of fatty acid synthesis through transcriptional regulation of the LDLR gene.

  13. GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling

    PubMed Central

    Lee, Elaine Choung-Hee

    2012-01-01

    Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is reduced dramatically by hypertonic stress or knockdown of rgpd genes encoding aminoacyl-tRNA synthetases and eukaryotic translation initiation factors (eIFs). Toxin-induced inhibition of translation also activates gpdh-1 expression. Hypertonicity-induced translation inhibition is mediated by general control nonderepressible (GCN)-2 kinase signaling and eIF-2α phosphoryation. Loss of gcn-1 or gcn-2 function prevents eIF-2α phosphorylation, completely blocks reductions in translation, and inhibits gpdh-1 transcription. gpdh-1 expression is regulated by the highly conserved with-no-lysine kinase (WNK) and Ste20 kinases WNK-1 and GCK-3, which function in the GCN-2 signaling pathway downstream from eIF-2α phosphorylation. Our previous work has shown that hypertonic stress causes rapid and dramatic protein damage in C. elegans and that inhibition of translation reduces this damage. The current studies demonstrate that reduced translation also serves as an essential signal for activation of WNK-1/GCK-3 kinase signaling and subsequent transcription of gpdh-1 and possibly other osmoprotective genes. PMID:23076791

  14. GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling.

    PubMed

    Lee, Elaine Choung-Hee; Strange, Kevin

    2012-12-15

    Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is reduced dramatically by hypertonic stress or knockdown of rgpd genes encoding aminoacyl-tRNA synthetases and eukaryotic translation initiation factors (eIFs). Toxin-induced inhibition of translation also activates gpdh-1 expression. Hypertonicity-induced translation inhibition is mediated by general control nonderepressible (GCN)-2 kinase signaling and eIF-2α phosphoryation. Loss of gcn-1 or gcn-2 function prevents eIF-2α phosphorylation, completely blocks reductions in translation, and inhibits gpdh-1 transcription. gpdh-1 expression is regulated by the highly conserved with-no-lysine kinase (WNK) and Ste20 kinases WNK-1 and GCK-3, which function in the GCN-2 signaling pathway downstream from eIF-2α phosphorylation. Our previous work has shown that hypertonic stress causes rapid and dramatic protein damage in C. elegans and that inhibition of translation reduces this damage. The current studies demonstrate that reduced translation also serves as an essential signal for activation of WNK-1/GCK-3 kinase signaling and subsequent transcription of gpdh-1 and possibly other osmoprotective genes.

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

    PubMed

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

    1998-02-20

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

  16. Aging-induced alterations in gene transcripts and functional activity of mitochondrial oxidative phosphorylation complexes in the heart.

    PubMed

    Preston, Claudia C; Oberlin, Andrew S; Holmuhamedov, Ekhson L; Gupta, Anu; Sagar, Sandeep; Syed, Rashad H Khazi; Siddiqui, Sabeeh A; Raghavakaimal, Sreekumar; Terzic, Andre; Jahangir, Arshad

    2008-06-01

    Aging is associated with progressive decline in energetic reserves compromising cardiac performance and tolerance to injury. Although deviations in mitochondrial functions have been documented in senescent heart, the molecular bases for the decline in energy metabolism are only partially understood. Here, high-throughput transcription profiles of genes coding for mitochondrial proteins in ventricles from adult (6-months) and aged (24-months) rats were compared using microarrays. Out of 614 genes encoding for mitochondrial proteins, 94 were differentially expressed with 95% downregulated in the aged. The majority of changes affected genes coding for proteins involved in oxidative phosphorylation (39), substrate metabolism (14) and tricarboxylic acid cycle (6). Compared to adult, gene expression changes in aged hearts translated into a reduced mitochondrial functional capacity, with decreased NADH-dehydrogenase and F(0)F(1) ATPase complex activities and capacity for oxygen-utilization and ATP synthesis. Expression of genes coding for transcription co-activator factors involved in the regulation of mitochondrial metabolism and biogenesis were downregulated in aged ventricles without reduction in mitochondrial density. Thus, aging induces a selective decline in activities of oxidative phosphorylation complexes I and V within a broader transcriptional downregulation of mitochondrial genes, providing a substrate for reduced energetic efficiency associated with senescence.

  17. Aging-Induced Alterations in Gene Transcripts and Functional Activity of Mitochondrial Oxidative Phosphorylation Complexes in the Heart

    PubMed Central

    Preston, Claudia C.; Oberlin, Andrew S.; Holmuhamedov, Ekhson L.; Gupta, Anu; Sagar, Sandeep; Khazi Syed, Rashad H.; Siddiqui, Sabeeh; Raghavakaimal, Sreekumar; Terzic, Andre; Jahangir, Arshad

    2008-01-01

    Aging is associated with progressive decline in energetic reserves compromising cardiac performance and tolerance to injury. Although deviations in mitochondrial functions have been documented in senescent heart, the molecular bases for the decline in energy metabolism are only partially understood. Here, high-throughput transcription profiles of genes coding for mitochondrial proteins in ventricles from adult (6-months) and aged (24-months) rats were compared using microarrays. Out of 614 genes encoding for mitochondrial proteins, 94 were differentially expressed with 95% downregulated in the aged. The majority of changes affected genes coding for proteins involved in oxidative phosphorylation (39), substrate metabolism (14) and tricarboxylic acid cycle (6). Compared to adult, gene expression changes in aged hearts translated into a reduced mitochondrial functional capacity, with decreased NADH-dehydrogenase and F0F1-ATPase complex activities and capacity for oxygen-utilization and ATP synthesis. Expression of genes coding for transcription co-activator factors involved in the regulation of mitochondrial metabolism and biogenesis were downregulated in aged ventricles without reduction in mitochondrial density. Thus, aging induces a selective decline in activities of oxidative phosphorylation complexes I and V within a broader transcriptional downregulation of mitochondrial genes, providing a substrate for reduced energetic efficiency associated with senescence. PMID:18400259

  18. Gene switching rate determines response to extrinsic perturbations in the self-activation transcriptional network motif

    PubMed Central

    de Franciscis, Sebastiano; Caravagna, Giulio; Mauri, Giancarlo; d’Onofrio, Alberto

    2016-01-01

    Gene switching dynamics is a major source of randomness in genetic networks, also in the case of large concentrations of the transcription factors. In this work, we consider a common network motif - the positive feedback of a transcription factor on its own synthesis - and assess its response to extrinsic noises perturbing gene deactivation in a variety of settings where the network might operate. These settings are representative of distinct cellular types, abundance of transcription factors and ratio between gene switching and protein synthesis rates. By investigating noise-induced transitions among the different network operative states, our results suggest that gene switching rates are key parameters to shape network response to external perturbations, and that such response depends on the particular biological setting, i.e. the characteristic time scales and protein abundance. These results might have implications on our understanding of irreversible transitions for noise-related phenomena such as cellular differentiation. In addition these evidences suggest to adopt the appropriate mathematical model of the network in order to analyze the system consistently to the reference biological setting. PMID:27256916

  19. Gene switching rate determines response to extrinsic perturbations in the self-activation transcriptional network motif.

    PubMed

    de Franciscis, Sebastiano; Caravagna, Giulio; Mauri, Giancarlo; d'Onofrio, Alberto

    2016-06-03

    Gene switching dynamics is a major source of randomness in genetic networks, also in the case of large concentrations of the transcription factors. In this work, we consider a common network motif - the positive feedback of a transcription factor on its own synthesis - and assess its response to extrinsic noises perturbing gene deactivation in a variety of settings where the network might operate. These settings are representative of distinct cellular types, abundance of transcription factors and ratio between gene switching and protein synthesis rates. By investigating noise-induced transitions among the different network operative states, our results suggest that gene switching rates are key parameters to shape network response to external perturbations, and that such response depends on the particular biological setting, i.e. the characteristic time scales and protein abundance. These results might have implications on our understanding of irreversible transitions for noise-related phenomena such as cellular differentiation. In addition these evidences suggest to adopt the appropriate mathematical model of the network in order to analyze the system consistently to the reference biological setting.

  20. Gene switching rate determines response to extrinsic perturbations in the self-activation transcriptional network motif

    NASA Astrophysics Data System (ADS)

    de Franciscis, Sebastiano; Caravagna, Giulio; Mauri, Giancarlo; D’Onofrio, Alberto

    2016-06-01

    Gene switching dynamics is a major source of randomness in genetic networks, also in the case of large concentrations of the transcription factors. In this work, we consider a common network motif - the positive feedback of a transcription factor on its own synthesis - and assess its response to extrinsic noises perturbing gene deactivation in a variety of settings where the network might operate. These settings are representative of distinct cellular types, abundance of transcription factors and ratio between gene switching and protein synthesis rates. By investigating noise-induced transitions among the different network operative states, our results suggest that gene switching rates are key parameters to shape network response to external perturbations, and that such response depends on the particular biological setting, i.e. the characteristic time scales and protein abundance. These results might have implications on our understanding of irreversible transitions for noise-related phenomena such as cellular differentiation. In addition these evidences suggest to adopt the appropriate mathematical model of the network in order to analyze the system consistently to the reference biological setting.

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

    PubMed Central

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

    2013-01-01

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

  2. A common transcriptional activator is located in the coding region of two replication-dependent mouse histone genes.

    PubMed Central

    Hurt, M M; Bowman, T L; Marzluff, W F

    1991-01-01

    There is a region in the mouse histone H3 gene protein-encoding sequence required for high expression. The 110-nucleotide coding region activating sequence (CRAS) from codons 58 to 93 of the H3.2 gene restored expression when placed 520 nucleotides 5' of the start of transcription in the correct orientation. Since identical mRNA molecules are produced by transcription of the original deletion gene and the deletion gene with the CRAS at -520, effects of the deletions on mRNA stability or other posttranscriptional events are completely ruled out. Inversion of the CRAS sequence in its proper position in the H3 gene resulted in only a threefold increase in expression, and placing the CRAS sequence 5' of the deleted gene in the wrong orientation had no effect on expression. In-frame deletions in the coding region of an H2a.2 gene led to identification of a 105-nucleotide sequence in the coding region between amino acids 50 and 85 necessary for high expression of the gene. Additionally, insertion of the H3 CRAS into the deleted region of the H2a.2 gene restored expression of the H2a gene. Thus, the CRAS element has an orientation-dependent, position-independent effect. Gel mobility shift competition studies indicate that the same proteins interact with both the H3 and H2a CRAS elements, suggesting that a common factor is involved in expression of histone genes. Images PMID:2038312

  3. The product of the murine homolog of the Drosophila extra sex combs gene displays transcriptional repressor activity.

    PubMed Central

    Denisenko, O N; Bomsztyk, K

    1997-01-01

    The heterogeneous nuclear ribonucleoprotein K protein represents a novel class of proteins that may act as docking platforms that orchestrate cross-talk among molecules involved in signal transduction and gene expression. Using a fragment of K protein as bait in the yeast two-hybrid screen, we isolated a cDNA that encodes a protein whose primary structure has extensive similarity to the Drosophila melanogaster extra sex combs (esc) gene product, Esc, a putative silencer of homeotic genes. The cDNA that we isolated is identical to the cDNA of the recently positionally cloned mouse embryonic ectoderm development gene, eed. Like Esc, Eed contains six WD-40 repeats in the C-terminal half of the protein and is thought to repress homeotic gene expression during mouse embryogenesis. Eed binds to K protein through a domain in its N terminus, but interestingly, this domain is not found in the Drosophila Esc. Gal4-Eed fusion protein represses transcription of a reporter gene driven by a promoter that contains Gal4-binding DNA elements. Eed also represses transcription when recruited to a target promoter by Gal4-K protein. Point mutations within the eed gene that are responsible for severe embryonic development abnormalities abolished the transcriptional repressor activity of Eed. Results of this study suggest that Eed-restricted homeotic gene expression during embryogenesis reflects the action of Eed as a transcriptional repressor. The Eed-mediated transcriptional effects are likely to reflect the interaction of Eed with multiple molecular partners, including K protein. PMID:9234727

  4. Retroviral insertions 90 kilobases proximal to the Evi-1 myeloid transforming gene activate transcription from the normal promoter.

    PubMed Central

    Bartholomew, C; Ihle, J N

    1991-01-01

    The inappropriate production of the Evi-1 zinc finger protein occurs in retrovirus-induced murine myeloid leukemias and human acute myelogenous leukemias. In murine leukemias, expression of the Evi-1 gene is associated with retroviral insertions either in the Evi-1 locus, which is immediately 5' of the coding region of the gene, or in the genetically linked Cb-1/fim-3 locus. In these studies, we demonstrate by chromosomal walking and pulse field electrophoresis that the Cb-1/fim-3 locus is located 90 kb 5' of the Evi-1 locus. Primary structure analysis of Evi-1 cDNA clones from a Cb-1/fim-3 rearranged cell line (DA-3) demonstrates that transcription initiates 5' of the Evi-1 locus and that the first noncoding exon of the gene is 681 bp larger than previously defined. S1 nuclease protection studies reveal multiple transcription initiation sites within this region. Comparable transcriptional initiation sites were identified in RNA from kidney and ovary, in which the gene is normally expressed, suggesting that retroviral insertions in the Cb-1/fim-3 locus activate transcription from the normal promoter. In one myeloid cell line (DA-3), a single long terminal repeat (LTR) is present in the Cb-1/fim-3 locus. No stable transcripts were detectable from this LTR. In cells with retroviral insertions in the Cb-1/fim-3 locus, one allele of the Evi-1 locus becomes hypermethylated in the 5' region of the gene. Together, these results are most consistent with an LTR-mediated, long-range cis activation of Evi-1 gene expression. Images PMID:1848663

  5. Sry is a transcriptional activator.

    PubMed

    Dubin, R A; Ostrer, H

    1994-09-01

    The SRY gene functions as a genetic switch in gonadal ridge initiating testis determination. The mouse Sry and human SRY open reading frames (ORFs) share a conserved DNA-binding domain (the HMG-box) yet exhibit no additional homology outside this region. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and mouse SRY ORFs contain a nuclear localization signal. The mouse Sry HMG-box domain selectively binds the sequence NACAAT in vitro when challenged with a random pool of oligonucleotides and binds AACAAT with the highest affinity. When put under the control of a heterologous promotor, the mouse Sry gene activated transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was likewise observed for a GAL4-responsive reporter gene, when the mouse Sry gene was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a glutamine/histidine-rich domain. In addition, LexA-mouse Sry fusion genes activated a LexA-responsive reporter gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and the mouse SRY ORFs encode nuclear, DNA-binding proteins and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

  6. Thyrotropin controls transcription of the thyroglobulin gene.

    PubMed

    Van Heuverswyn, B; Streydio, C; Brocas, H; Refetoff, S; Dumont, J; Vassart, G

    1984-10-01

    The availability of rat thyroglobulin cDNA clones was exploited to study the regulation of thyroglobulin gene transcription by thyrotropin (TSH). Groups of rats were subjected to treatments leading to reduction or increase in the rat serum TSH (rTSH) levels. Thyroid gland nuclei were isolated, incubated in vitro in the presence of 32P-labeled uridine triphosphate, and thyroglobulin transcripts were quantitated by hybridization to immobilized rat thyroglobulin cDNA clones. Transcription of the thyroglobulin gene was found to be very active in thyroid nuclei from control animals. It represented about 10% of total RNA polymerase II activity. Chronic hyperstimulation of the thyroid glands with endogenous rTSH was achieved in rats treated with the goitrogen propylthiouracil. No significant increase of thyroglobulin gene transcription could be measured in thyroid nuclei from these animals. On the contrary, a dramatic decrease in thyroglobulin gene transcription was observed in those animals in which endogenous rTSH levels had been suppressed by hypophysectomy or by the administration of triiodothyronine. Injection of exogenous bovine TSH in such animals readily restored transcriptional activity of the gene. Our results identify transcription as an important regulatory step involved in TSH action. They suggest that normal TSH levels induce close to maximal expression of the thyroglobulin gene but that continuous presence of TSH is required in order to maintain the gene in an activated state.

  7. Transcriptional activation of TFEB/ZKSCAN3 target genes underlies enhanced autophagy in spinobulbar muscular atrophy.

    PubMed

    Chua, Jason P; Reddy, Satya L; Merry, Diane E; Adachi, Hiroaki; Katsuno, Masahisa; Sobue, Gen; Robins, Diane M; Lieberman, Andrew P

    2014-03-01

    Spinobulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in exon 1 of the androgen receptor (AR) gene. SBMA demonstrates androgen-dependent toxicity due to unfolding and aggregation of the mutant protein. There are currently no disease-modifying therapies, but of increasing interest for therapeutic targeting is autophagy, a highly conserved cellular process mediating protein quality control. We have previously shown that genetic manipulations inhibiting autophagy diminish skeletal muscle atrophy and extend the lifespan of AR113Q knock-in mice. In contrast, manipulations inducing autophagy worsen muscle atrophy, suggesting that chronic, aberrant upregulation of autophagy contributes to pathogenesis. Since the degree to which autophagy is altered in SBMA and the mechanisms responsible for such alterations are incompletely defined, we sought to delineate autophagic status in SBMA using both cellular and mouse models. Here, we confirm that autophagy is induced in cellular and knock-in mouse models of SBMA and show that the transcription factors transcription factor EB (TFEB) and ZKSCAN3 operate in opposing roles to underlie these changes. We demonstrate upregulation of TFEB target genes in skeletal muscle from AR113Q male mice and SBMA patients. Furthermore, we observe a greater response in AR113Q mice to physiological stimulation of autophagy by both nutrient starvation and exercise. Taken together, our results indicate that transcriptional signaling contributes to autophagic dysregulation and provides a mechanistic framework for the pathologic increase of autophagic responsiveness in SBMA.

  8. Transcriptional activities of the Pax6 gene eyeless regulate tissue specificity of ectopic eye formation in Drosophila

    PubMed Central

    Weasner, Bonnie M.; Weasner, Brandon; DeYoung, Stephanie M.; Michaels, Scott D.; Kumar, Justin P.

    2009-01-01

    Pax genes encode DNA binding proteins that play pivotal roles in the determination of complex tissues. Members of one subclass, Pax6, function as selector genes and play key roles in the retinal development of all seeing animals. Mutations within the Pax6 homologs including fly eyeless, mouse Small eye and human Pax6 lead to severe retinal defects in their respective systems. In Drosophila eyeless and twin of eyeless, play non-redundant roles in the developing retina. One particularly interesting characteristic of these genes is that, although expression of either gene can induce ectopic eye formation in non-retinal tissues, there are differences in the location and frequencies at which the eyes develop. eyeless induces much larger ectopic eyes, at higher frequencies, and in a broader range of tissues than twin of eyeless. In this report we describe a series of experiments conducted in both yeast and flies that has identified protein modules that are responsible for the differences in tissue transformation. These domains appear to contain transcriptional activator and repressor activity of distinct strengths. We propose a model in which the selective presence of these activities and their relative strengths accounts, in part, for the disparity to which ectopic eyes are induced in response to the forced expression of eyeless and twin of eyeless. The identification of both transcriptional activator and repressor activity within the Pax6 protein furthers our understanding of how this gene family regulates tissue determination. PMID:19406113

  9. Deduction of upstream sequences of Xanthomonas campestris flagellar genes responding to transcription activation by FleQ

    SciTech Connect

    Hu, R.-M.; Yang, T.-C.; Yang, S.-H.; Tseng, Y.-H. . E-mail: yhtseng@chtai.ctc.edu.tw

    2005-10-07

    Xanthomonas campestris pv. campestris (Xcc), a close relative to Pseudomonas aeruginosa, is the pathogen causing black rot in cruciferous plants. In P. aeruginosa, FleQ serves as a cognate activator of {sigma}{sup 54} in transcription from several {sigma}{sup 54}-dependent promoters of flagellar genes. These P. aeruginosa promoters have been analyzed for FleQ-binding sequences; however, no consensus was deduced. Xcc, although lacks fleSR, has a fleQ homologue residing among over 40 contiguously clustered flagellar genes. A fleQ mutant, Xc17fleQ, constructed by insertional mutation is deficient in FleQ protein, non-flagellated, and immobile. Transcriptional fusion assays on six putative {sigma}{sup 54}-dependent promoters of the flagellar genes, fliE, fliQ, fliL, flgG, flgB, and flhF, indicated that each of them is also FleQ dependent. Each of these promoters has a sequence with weak consensus to 5'-gaaacCCgccgCcgctTt-3', immediately upstream of the predicted {sigma}{sup 54}-binding site, with an imperfect inverted repeat containing a GC-rich center flanked by several A and T at 5'- and 3'-ends, respectively. Replacing this region in fliE promoter with a HindIII recognition sequence abolished the transcription, indicating that this region responds to transcription activation by FleQ.

  10. Induction of human adiponectin gene transcription by telmisartan, angiotensin receptor blocker, independently on PPAR-{gamma} activation

    SciTech Connect

    Moriuchi, Akie ||. E-mail: f1195@cc.nagasaki-u-ac.jp; Shimamura, Mika; Kita, Atsushi; Kuwahara, Hironaga; Satoh, Tsuyoshi; Satoh, Tsuyoshi; Fujishima, Keiichiro; Fukushima, Keiko |; Hayakawa, Takao; Mizuguchi, Hiroyuki; Nagayama, Yuji; Kawasaki, Eiji

    2007-05-18

    Adiponectin, an adipose tissue-specific plasma protein, has been shown to ameliorate insulin resistance and inhibit the process of atherosclerosis. Recently, several reports have stated that angiotensin type 1 receptor blockers (ARBs), increase adiponectin plasma level, and ameliorate insulin resistance. Telmisartan, a subclass of ARBs, has been shown to be a partial agonist of the peroxisome proliferator-activated receptor (PPAR)-{gamma}, and to increase the plasma adiponectin level. However, the transcriptional regulation of the human adiponectin gene by telmisartan has not been determined yet. To elucidate the effect of telmisartan on adiponectin, the stimulatory regulation of human adiponectin gene by telmisartan was investigated in 3T3-L1 adipocytes, utilizing adenovirus-mediated luciferase reporter gene-transferring technique. This study indicates that telmisartan may stimulate adiponectin transcription independent of PPAR-{gamma}.

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

    PubMed

    Mahajan, Muktar A; Stanley, Frederick M

    2014-01-25

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

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

    PubMed Central

    Virbasius, J V; Scarpulla, R C

    1994-01-01

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

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

    EPA Science Inventory

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

  14. Cystatin D Locates in the Nucleus at Sites of Active Transcription and Modulates Gene and Protein Expression*

    PubMed Central

    Ferrer-Mayorga, Gemma; Alvarez-Díaz, Silvia; Valle, Noelia; De Las Rivas, Javier; Mendes, Marta; Barderas, Rodrigo; Canals, Francesc; Tapia, Olga; Casal, J. Ignacio; Lafarga, Miguel; Muñoz, Alberto

    2015-01-01

    Cystatin D is an inhibitor of lysosomal and secreted cysteine proteases. Strikingly, cystatin D has been found to inhibit proliferation, migration, and invasion of colon carcinoma cells indicating tumor suppressor activity that is unrelated to protease inhibition. Here, we demonstrate that a proportion of cystatin D locates within the cell nucleus at specific transcriptionally active chromatin sites. Consistently, transcriptomic analysis show that cystatin D alters gene expression, including that of genes encoding transcription factors such as RUNX1, RUNX2, and MEF2C in HCT116 cells. In concordance with transcriptomic data, quantitative proteomic analysis identified 292 proteins differentially expressed in cystatin D-expressing cells involved in cell adhesion, cytoskeleton, and RNA synthesis and processing. Furthermore, using cytokine arrays we found that cystatin D reduces the secretion of several protumor cytokines such as fibroblast growth factor-4, CX3CL1/fractalkine, neurotrophin 4 oncostatin-M, pulmonary and activation-regulated chemokine/CCL18, and transforming growth factor B3. These results support an unanticipated role of cystatin D in the cell nucleus, controlling the transcription of specific genes involved in crucial cellular functions, which may mediate its protective action in colon cancer. PMID:26364852

  15. The full-length transcript of a caulimovirus is a polycistronic mRNA whose genes are trans activated by the product of gene VI.

    PubMed

    Scholthof, H B; Gowda, S; Wu, F C; Shepherd, R J

    1992-05-01

    Gene expression of figwort mosaic virus (FMV), a caulimovirus, was investigated by electroporation of Nicotiana edwardsonii cell suspension protoplasts with cloned viral constructs in which a reporter gene was inserted at various positions on the genome. The results showed that the genome of FMV contains two promoters; one is used for the production of a full-length RNA and another initiates synthesis of a separate monocistronic RNA for gene VI. Evidence is provided that the full-length transcript, the probable template for reverse transcription, can serve as a polycistronic mRNA for translation of genes I through V and perhaps also gene VI. Expression of all the genes on the polycistronic mRNA is trans activated by the gene VI protein. Reporter gene expression appears most efficient when its start codon is in close proximity to the stop codon of the preceding gene, as for the native genes of caulimoviruses. We propose that the gene VI product enables expression of the polycistronic mRNA by promoting reinitiation of ribosomes to give translational coupling of individual genes.

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

    PubMed Central

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

    2007-01-01

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

  17. The Hansenula polymorpha MOX gene presents two alternative transcription start points differentially utilized and sensitive to respiratory activity.

    PubMed

    Genu, Victor; Gödecke, Stefanie; Hollenberg, Cornelis P; Pereira, Gonçalo G

    2003-06-01

    The peroxisomal methanol metabolism of Hansenula polymorpha depends on a group of genes that are coordinately regulated. Methanol oxidase (Mox) plays a key role in this pathway and its synthesis has been shown to be regulated at the transcriptional level. MOX expression is strongly repressed on glucose and activated on glycerol or methanol. In this study we have identified two MOX transcripts that are differentially expressed along MOX derepression. The first one, named l-MOX (for longer MOX), starts at position -425, is only weakly and transiently transcribed and is not translated into the Mox protein. The other is the true MOX mRNA, which initiates around position -25. Using a strain bearing multiple copies of MOX(Q1N) and a reporter gene fused to the MOX promoter, regulation of the two transcripts was investigated. Initiation of the true MOX correlates with repression of l-MOX and conditions that are repressive for MOX transcription, such as the inhibition of mitochondrial activity, lead to higher levels of l-MOX expression. This effect was first observed in a mox mutant (Q1N-M8) unable to grow on nonfermentable carbon sources. No function was detected for l-MOX, but its regulation follows a pattern similar to that of catalase, which is essential for methanol metabolism. This suggests that, l-MOX, although precisely regulated, seems to be a remnant of the evolution of the methanol metabolism network.

  18. Analysis of Transcriptionally Active Gene Clusters of Major Outer Membrane Protein Multigene Family in Ehrlichia canis and E. chaffeensis

    PubMed Central

    Ohashi, Norio; Rikihisa, Yasuko; Unver, Ahmet

    2001-01-01

    Ehrlichia canis and E. chaffeensis are tick-borne obligatory intramonocytic ehrlichiae that cause febrile systemic illness in humans and dogs, respectively. The current study analyzed the pleomorphic multigene family encoding approximately 30-kDa major outer membrane proteins (OMPs) of E. canis and E. chaffeensis. Upstream from secA and downstream of hypothetical transcriptional regulator, 22 paralogs of the omp gene family were found to be tandemly arranged except for one or two genes with opposite orientations in a 28- and a 27-kb locus in the E. canis and E. chaffeensis genomes, respectively. Each locus consisted of three highly repetitive regions with four nonrepetitive intervening regions. E. canis, in addition, had a 6.9-kb locus which contained a repeat of three tandem paralogs in the 28-kb locus. These total 47 paralogous and orthologous genes encoded OMPs of approximately 30 to 35 kDa consisting of several hypervariable regions alternating with conserved regions. In the 5′-end half of the 27-kb locus or the 28-kb locus of each Ehrlichia species, 14 paralogs were linked by short intergenic spaces ranging from −8 bp (overlapped) to 27 bp, and 8 remaining paralogs in the 3′-end half were connected by longer intergenic spaces ranging from 213 to 632 bp. All 22 paralogs, five unknown genes, and secA in the omp cluster in E. canis were transcriptionally active in the monocyte culture, and the paralogs with short intergenic spaces were cotranscribed with their adjacent genes, including the respective intergenic spaces at both the 5′ and the 3′ sides. Although omp genes are diverse, our results suggest that the gene organization of the clusters and the gene locus are conserved between two species of Ehrlichia to maintain a unique transcriptional mechanism for adaptation to environmental changes common to them. PMID:11254561

  19. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression.

    PubMed Central

    Gambello, M J; Iglewski, B H

    1991-01-01

    We report the discovery of the lasR gene, which positively regulates elastase expression in Pseudomonas aeruginosa PAO1. The lasR gene was cloned by its ability to restore a positive elastase phenotype in strain PA103, a strain which possesses the elastase structural gene (lasB) but fails to synthesize the enzyme. Nucleotide sequence analysis revealed an open reading frame of 716 nucleotides encoding a protein of approximately 27 kDa. A labeled LasR protein of 27 kDa was detected in Escherichia coli by using a T7 RNA polymerase expression system. A chromosomal deletion mutant of the lasR gene was constructed in PAO1 by gene replacement. This mutant (PAO-R1) is devoid of elastolytic activity and elastase antigen. The deduced amino acid sequence of LasR is 27% homologous to the positive activator LuxR of Vibrio fischeri and the suspected activator 28K-UvrC of E. coli. Northern (RNA) analysis of total cellular RNA from PAO1, PAO-R1, and PAO-R1 containing the lasR gene on a multicopy plasmid (pMG1.7) revealed that a functional lasR gene is required for transcription of the elastase structural gene (lasB). Images PMID:1902216

  20. A novel PRD I and TG binding activity involved in virus-induced transcription of IFN-A genes.

    PubMed Central

    Génin, P; Bragança, J; Darracq, N; Doly, J; Civas, A

    1995-01-01

    Comparative analysis of the inducible elements of the mouse interferon A4 and A11 gene promoters (IE-A4 and IE-A11) by transient transfection experiments, DNase 1 footprinting and electrophoretic mobility shift assays resulted in identification of a virus-induced binding activity suggested to be involved in NDV-induced activation of transcription of these genes. The virus-induced factor, termed VIF, is activated early by contact of virions with cells. It specifically recognizes the PRD I-like domain shared by both inducible elements, as well as the TG-like domain of IE-A4. This factor, distinct from the IRF-1, IRF-2 and the alpha F1 binding proteins and presenting a different affinity pattern from that of the TG protein, is proposed as a candidate for IFN-type I gene regulation. Images PMID:8559665

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

    PubMed

    Meier, Andreas; Thoma, Fritz

    2005-03-01

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

  2. Vibrio parahaemolyticus CalR down regulates the thermostable direct hemolysin (TDH) gene transcription and thereby inhibits hemolytic activity.

    PubMed

    Zhang, Yiquan; Zhang, Ying; Gao, He; Zhang, Lingyu; Yin, Zhe; Huang, Xinxiang; Zhou, Dongsheng; Yang, Huiying; Yang, Wenhui; Wang, Li

    2017-03-04

    TDH, encoded by tdh gene, is a major virulent determinant of V. parahaemolyticus that controls various biological activities, such as hemolytic activity, cytotoxicity, and enterotoxicity. The hemolytic activity on Wagatsuma agar ascribed to TDH is called Kanagawa phenomenon (KP). All KP positive strains contain tdh1 and tdh2 genes, but tdh2 is predominantly responsible for KP. CalR is a regulatory protein that was originally identified as a repressor of swarming motility and T3SS1 gene expression in V. parahaemolyticus. In the present study, the regulation of tdh2 by CalR was investigated using a set of experiments including qRT-PCR, primer extension, LacZ fusion, hemolytic phenotype, EMSA, and DNase I footprinting assays. The results showed that His-CalR protected a single region from 224bp to 318bp upstream of tdh2 against DNase I digestion, and a transcriptional start site located at 42bp upstream of tdh2 was detected and its transcribed activity was inhibited by CalR. Moreover, the KP test results showed that the hemolytic activity of V. parahaemolyticus is also under negative control of CalR. The data demonstrated that CalR is a repressor of the tdh2 transcription and thereby inhibits the hemolytic activity of V. parahaemolyticus.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-04-01

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

  6. DNA damage induces N-acetyltransferase NAT10 gene expression through transcriptional activation.

    PubMed

    Liu, Haijing; Ling, Yun; Gong, Yilei; Sun, Ying; Hou, Lin; Zhang, Bo

    2007-06-01

    NAT10 (N-acetyltransferase 10) is a protein with histone acetylation activity and primarily identified to be involved in regulation of telomerase activity. The presented research shows its transcriptional activation by genotoxic agents and possible role in DNA damage. NAT10 mRNA could be markedly increased by using hydrogen peroxide (H2O2) or cisplatin in a dose- and time-dependent way, and the immunofluorescent staining revealed that the treatment of H2O2 or cisplatin induced focal accumulation of NAT10 protein in cellular nuclei. Both H2O2 and cisplatin could stimulate the transcriptional activity of the NAT10 promoter through the upstream sequences from -615 bp to +110 bp, with which some nuclear proteins interacted. Ectopic expression of NAT10 could enhance the number of survival cells in the presence of H2O2 or cisplatin. The above results suggested that NAT10 could be involved in DNA damage response and increased cellular resistance to genotoxicity.

  7. Widespread Inducible Transcription Downstream of Human Genes

    PubMed Central

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

    2015-01-01

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

  8. Signal transducer and activator of transcription (STAT)-3 regulates microRNA gene expression in chronic lymphocytic leukemia cells

    PubMed Central

    2013-01-01

    Backgrounds Approximately 1,000 microRNAs (miRs) are present in the human genome; however, little is known about the regulation of miR transcription. Because miR levels are deregulated in chronic lymphocytic leukemia (CLL) and signal transducer and activator of transcription (STAT)-3 is constitutively activated in CLL, we sought to determine whether STAT3 affects the transcription of miR genes in CLL cells. Methods We used publically available data from the ENCODE project to identify putative STAT3 binding sites in the promoters of miR genes. Then we transfected CLL cells with STAT3-shRNA or with an empty vector, and to determine which miRs are differentially expressed, we used a miR microarray approach followed by validation of the microarray results for 6 miRs using quantitative real-time polymerase chain reaction (qRT-PCR). Results We identified putative STAT3 binding sites in 160 promoter regions of 200 miRs, including miR-21, miR-29, and miR-155, whose levels have been reported to be upregulated in CLL. Levels of 72 miRs were downregulated (n = 63) or upregulated (n = 9). qRT-PCR confirmed the array data in 5 of 6 miRs. Conclusions The presence of activated STAT3 has a profound effect on miR expression in CLL cells. PMID:23725032

  9. Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation.

    PubMed Central

    Carrier, F; Owens, R A; Nebert, D W; Puga, A

    1992-01-01

    Transcriptional activation of the murine Cyp1a-1 (cytochrome P(1)450) gene by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (dioxin) requires the aromatic hydrocarbon (Ah) receptor and the interaction of an inducer-receptor complex with one or more of the Ah-responsive elements (AhREs) located about 1 kb upstream from the transcriptional initiation site. We find that treatment of mouse hepatoma Hepa-1 cells with 2-aminopurine, an inhibitor of protein kinase activity, inhibits CYP1A1 mRNA induction by TCDD as well as the concomitant increase in CYP1A1 enzyme activity. Formation of DNA-protein complexes between the Ah receptor and its AhRE target is also inhibited by 2-aminopurine, as determined by gel mobility shift assays. Phosphorylation is required for the formation of Ah receptor-specific complexes, since in vitro dephosphorylation of nuclear extracts from TCDD-treated Hepa-1 cells abolishes the capacity of the Ah receptor to form specific complexes with its cognate AhRE sequences. To determine whether any one of several known protein kinases was involved in the transcriptional regulation of the Cyp1a-1 gene, we treated Hepa-1 cells with nine other protein kinase inhibitors prior to induction with TCDD; nuclear extracts from these cells were analyzed for their capacity to form specific DNA-protein complexes. Only extracts from cells treated with staurosporine, a protein kinase C inhibitor, were unable to form these complexes. In addition, staurosporine completely inhibited CYP1A1 mRNA induction by TCDD. Depletion of protein kinase C by prolonged treatment with phorbol ester led to the complete suppression of CYP1A1 mRNA induction by TCDD. We conclude that (i) phosphorylation is necessary for the formation of a transcriptional complex and for transcriptional activation of the Cyp1a-1 gene; (ii) the phosphorylation site(s) exists on at least one of the proteins constituting the transcriptional complex, possibly the Ah receptor itself; and (iii) the

  10. The bacteriophage-derived transcriptional regulator, LscR, activates the expression of levansucrase genes in Pseudomonas syringae.

    PubMed

    Abdallah, Khaled; Hartman, Katharina; Pletzer, Daniel; Zhurina, Daria; Ullrich, Matthias S

    2016-12-01

    Synthesis of the exopolysaccharide levan occurs in the bacterial blight pathogen of soybean, Pseudomonas syringae pv. glycinea PG4180, when this bacterium encounters moderate to high concentrations of sucrose inside its host plant. The process is mediated by the temperature-dependent expression and secretion of two levansucrases, LscB and LscC. Previous studies showed the importance of a prophage-associated promoter element in driving the expression of levansucrase genes. Herein, heterologous screening for transcriptional activators revealed that the prophage-borne transcriptional regulator, LscR, from P. syringae mediates expression of levansucrase. A lscR-deficient mutant was generated and exhibited a levan-negative phenotype when grown on a sucrose-rich medium. This phenotype was confirmed by zymographic analysis and Western blots which demonstrated absence of levansucrase in the supernatant and total cell lysates. Transcriptional analysis showed a down-regulation of expression levels of levansucrase and glycosyl hydrolase genes in the lscR-deficient mutant. Ultimately, a direct binding of LscR to the promoter region of levansucrase was demonstrated using electrophoretic mobility shift assays allowing to conclude that a bacteriophage-derived regulator dictates expression of bacterial genes involved in in planta fitness.

  11. The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF.

    PubMed

    Niu, Xiaolei; Zhou, Meiliang; Henkel, Christiaan V; van Heusden, G Paul H; Hooykaas, Paul J J

    2015-12-01

    During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A. thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process.

  12. Cell density-dependent transcriptional activation of endocrine-related genes in human adipose tissue-derived stem cells.

    PubMed

    Ghosh, Sagar; Dean, Angela; Walter, Marc; Bao, Yongde; Hu, Yanfen; Ruan, Jianhua; Li, Rong

    2010-08-01

    Adipose tissue is recognized as an endocrine organ that plays an important role in human diseases such as type II diabetes and cancer. Human adipose tissue-derived stem cells (ASCs), a distinct cell population in adipose tissue, are capable of differentiating into multiple lineages including adipogenesis. When cultured in vitro under a confluent condition, ASCs reach a commitment stage for adipogenesis, which can be further induced into terminally differentiated adipocytes by a cocktail of adipogenic factors. Here we report that the confluent state of ASCs triggers transcriptional activation cascades for genes that are responsible for the endocrine function of adipose tissue. These include insulin-like growth factor 1 (IGF-1) and aromatase (Cyp19), a key enzyme in estrogen biosynthesis. Despite similar adipogenic potentials, ASCs from different individuals display huge variations in activation of these endocrine-related genes. Bioinformatics and experimental data suggest that transcription factor Foxo1 controls a large number of "early" confluency-response genes, which subsequently induce "late" response genes. Furthermore, siRNA-mediated knockdown of Foxo1 substantially compromises the ability of committed ASCs to stimulate tumor cell migration in vitro. Thus, our work suggests that cell density is an important determinant of the endocrine potential of ASCs.

  13. Transcriptional activity of novel ALDH1L1 promoters in the rat brain following AAV vector-mediated gene transfer

    PubMed Central

    Mudannayake, Janitha M; Mouravlev, Alexandre; Fong, Dahna M; Young, Deborah

    2016-01-01

    Aldehyde dehydrogenase family 1, member L1 (ALDH1L1) is a recently characterized pan-astrocytic marker that is more homogenously expressed throughout the brain than the classic astrocytic marker, glial fibrillary acidic protein. We generated putative promoter sequence variants of the rat ALDH1L1 gene for use in adeno-associated viral vector-mediated gene transfer, with an aim to achieve selective regulation of transgene expression in astrocytes in the rat brain. Unexpectedly, ALDH1L1 promoter variants mediated transcriptional activity exclusively in neurons in the substantia nigra pars compacta as assessed by luciferase reporter expression at 3 weeks postvector infusion. This selectivity for neurons in the substantia nigra pars compacta also persisted in the context of adeno-associated viral serotype 5, 8 or 9 vector-mediated gene delivery. An in vivo promoter comparison showed the highest performing ALDH1L1 promoter variant mediated higher transgene expression than the neuronal-specific synapsin 1 and tyrosine hydroxylase promoters. The ALDH1L1 promoter was also transcriptionally active in dentate granule neurons following intrahippocampal adeno-associated viral vector infusion, whereas transgene expression was detected in both striatal neurons and astrocytes following vector infusion into the striatum. Our results demonstrate the potential suitability of the ALDH1L1 promoter as a new tool in the development of gene therapy and disease modelling applications. PMID:27990448

  14. A novel peroxisome proliferator response element modulates hepatic low-density lipoprotein receptor gene transcription in response to PPARδ activation.

    PubMed

    Shende, Vikram R; Singh, Amar Bahadur; Liu, Jingwen

    2015-12-15

    The hepatic expression of low-density lipoprotein (LDL) receptor (LDLR) gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative peroxisome proliferator-activated receptor (PPAR)-response element (PPRE) sequence motif located at -768 to -752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin (RSV)-mediated transactivation. EMSA and ChIP assay further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression.

  15. Identification and characterization of the spiruchostatin biosynthetic gene cluster enables yield improvement by overexpressing a transcriptional activator

    PubMed Central

    Potharla, Vishwakanth Y.; Wang, Cheng; Cheng, Yi-Qiang

    2014-01-01

    Spiruchostatins A and B are members of the FK228-family of natural products with potent histone deacetylase inhibitory activities and antineoplastic activities. However, their production in the wild-type strain of Pseudomonas sp. Q71576 is low. To improve the yield, the spiruchostatin biosynthetic gene cluster (spi) was first identified by rapid genome sequencing and characterized by genetic mutations. This spi gene cluster encodes a hybrid biosynthetic pathway similar to that encoded by the FK228 biosynthetic gene cluster (dep) in Chromobacterium violaceum No. 968. Each gene cluster contains a pathway regulatory gene (spiR vs. depR) but these two genes encode transcriptional activators of different classes. Overexpression of native spiR or heterologous depR in the wild-type strain of Pseudomonas sp. Q71576 resulted in 268% or 1,285% increase of the combined titer of spiruchostatins A and B, respectively. RT-PCR analysis indicates that overexpression of heterologous depR upregulates the expression of native spiR. PMID:24973954

  16. Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and brown adipocyte differentiation.

    PubMed Central

    Schlüter, Agatha; Barberá, Maria José; Iglesias, Roser; Giralt, Marta; Villarroya, Francesc

    2002-01-01

    Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5' enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis. PMID:11829740

  17. A Novel Peroxisome Proliferator Response Element Modulates Hepatic Low Density Lipoprotein Receptor Gene Transcription in Response to PPARδ Activation

    PubMed Central

    Shende, Vikram R.; Singh, Amar Bahadur; Liu, Jingwen

    2016-01-01

    The hepatic expression of LDLR gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative PPAR-response element (PPRE) sequence motif located at −768 to −752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin mediated transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression. PMID:26443862

  18. Bordetella pertussis fim3 gene regulation by BvgA: phosphorylation controls the formation of inactive vs. active transcription complexes.

    PubMed

    Boulanger, Alice; Moon, Kyung; Decker, Kimberly B; Chen, Qing; Knipling, Leslie; Stibitz, Scott; Hinton, Deborah M

    2015-02-10

    Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at P(fim3). However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates P(fim3) activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating P(fim3) activation once the signal for BvgA phosphorylation is removed.

  19. Transcriptional gene silencing in humans

    PubMed Central

    Weinberg, Marc S.; Morris, Kevin V.

    2016-01-01

    It has been over a decade since the first observation that small non-coding RNAs can functionally modulate epigenetic states in human cells to achieve functional transcriptional gene silencing (TGS). TGS is mechanistically distinct from the RNA interference (RNAi) gene-silencing pathway. TGS can result in long-term stable epigenetic modifications to gene expression that can be passed on to daughter cells during cell division, whereas RNAi does not. Early studies of TGS have been largely overlooked, overshadowed by subsequent discoveries of small RNA-directed post-TGS and RNAi. A reappraisal of early work has been brought about by recent findings in human cells where endogenous long non-coding RNAs function to regulate the epigenome. There are distinct and common overlaps between the proteins involved in small and long non-coding RNA transcriptional regulatory mechanisms, suggesting that the early studies using small non-coding RNAs to modulate transcription were making use of a previously unrecognized endogenous mechanism of RNA-directed gene regulation. Here we review how non-coding RNA plays a role in regulation of transcription and epigenetic gene silencing in human cells by revisiting these earlier studies and the mechanistic insights gained to date. We also provide a list of mammalian genes that have been shown to be transcriptionally regulated by non-coding RNAs. Lastly, we explore how TGS may serve as the basis for development of future therapeutic agents. PMID:27060137

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

    PubMed

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

    2012-01-01

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

  1. The anti-tumor drug bleomycin preferentially cleaves at the transcription start sites of actively transcribed genes in human cells.

    PubMed

    Murray, Vincent; Chen, Jon K; Galea, Anne M

    2014-04-01

    The genome-wide pattern of DNA cleavage at transcription start sites (TSSs) for the anti-tumor drug bleomycin was examined in human HeLa cells using next-generation DNA sequencing. It was found that actively transcribed genes were preferentially cleaved compared with non-transcribed genes. The 143,600 identified human TSSs were split into non-transcribed genes (82,596) and transcribed genes (61,004) for HeLa cells. These transcribed genes were further split into quintiles of 12,201 genes comprising the top 20, 20-40, 40-60, 60-80, and 80-100 % of expressed genes. The bleomycin cleavage pattern at highly transcribed gene TSSs was greatly enhanced compared with purified DNA and non-transcribed gene TSSs. The top 20 and 20-40 % quintiles had a very similar enhanced cleavage pattern, the 40-60 % quintile was intermediate, while the 60-80 and 80-100 % quintiles were close to the non-transcribed and purified DNA profiles. The pattern of bleomycin enhanced cleavage had peaks that were approximately 200 bp apart, and this indicated that bleomycin was identifying the presence of phased nucleosomes at TSSs. Hence bleomycin can be utilized to detect chromatin structures that are present at actively transcribed genes. In this study, for the first time, the pattern of DNA damage by a clinically utilized cancer chemotherapeutic agent was performed on a human genome-wide scale at the nucleotide level.

  2. Cryptic carbapenem antibiotic production genes are widespread in Erwinia carotovora: facile trans activation by the carR transcriptional regulator.

    PubMed

    Holden, M T; McGowan, S J; Bycroft, B W; Stewart, G S; Williams, P; Salmond, G P

    1998-06-01

    Few strains of Erwinia carotovora subsp. carotovora (Ecc) make carbapenem antibiotics. Strain GS101 makes the basic carbapenem molecule, 1-carbapen-2-em-3-carboxylic acid (Car). The production of this antibiotic has been shown to be cell density dependent, requiring the accumulation of the small diffusible molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) in the growth medium. When the concentration of this inducer rises above a threshold level, OHHL is proposed to interact with the transcriptional activator of the carbapenem cluster (CarR) and induce carbapenem biosynthesis. The introduction of the GS101 carR gene into an Ecc strain (SCRI 193) which is naturally carbapenem-negative resulted in the production of Car. This suggested that strain SCRI 193 contained functional cryptic carbapenem biosynthetic genes, but lacked a functional carR homologue. The distribution of trans-activatable antibiotic genes was assayed in Erwinia strains from a culture collection and was found to be common in a large proportion of Ecc strains. Significantly, amongst the Ecc strains identified, a larger proportion contained trans-activatable cryptic genes than produced antibiotics constitutively. Southern hybridization of the chromosomal DNA of cryptic Ecc strains confirmed the presence of both the car biosynthetic cluster and the regulatory genes. Identification of homologues of the transcriptional activator carR suggests that the cause of the silencing of the carbapenem biosynthetic cluster in these strains is not the deletion of carR. In an attempt to identify the cause of the silencing in the Ecc strain SCRI 193 the carR homologue from this strain was cloned and sequenced. The SCRI 193 CarR homologue was 94% identical to the GS101 CarR and contained 14 amino acid substitutions. Both homologues could be expressed from their native promoters and ribosome-binding sites using an in vitro prokaryotic transcription and translation assay, and when the SCRI 193 carR homologue was

  3. Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c2 Gene P2 Promoter by the Response Regulator PrrA

    PubMed Central

    Comolli, James C.; Carl, Audrey J.; Hall, Christine; Donohue, Timothy

    2002-01-01

    Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the α subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c2 gene (cycA) promoter, P2, which contained sequences from −73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo. PMID:11751815

  4. CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes.

    PubMed

    Ogiwara, Hideaki; Kohno, Takashi

    2012-01-01

    Histone acetylation at DNA double-strand break (DSB) sites by CBP and p300 histone acetyltransferases (HATs) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the BRCA1 and RAD51 genes, which are involved in homologous recombination (HR), a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.

  5. The dose dependence of glucocorticoid-inducible gene expression results from changes in the number of transcriptionally active templates.

    PubMed Central

    Ko, M S; Nakauchi, H; Takahashi, N

    1990-01-01

    Glucocorticoid hormones induce the transcription of genes having glucocorticoid response elements in a dose dependent manner. To determine whether this dose dependence represents a response of individual templates or of the mass of templates, we introduced a bacterial beta-galactosidase gene linked to the glucocorticoid-inducible enhancer/promoter of the mouse mammary tumor virus (MTV) into Ltk- cells and obtained stable transformants containing a single or a few templates per cell. Visual inspection and flow cytometry analysis by enzyme histochemistry assay for beta-galactosidase revealed that individual cells showed very heterogeneous beta-galactosidase activity after 48 h induction with dexamethasone. When the glucocorticoid concentration was increased, an increasing cell population producing beta-galactosidase was observed. These phenomena were probably not due to heterogeneity of template copy number or to a predetermined cellular state among individual cells, since cells forming a single small colony gave similar results. This was also supported by data showing that recloned cells retained both their responsiveness to the glucocorticoid hormone and their digestion pattern in Southern blotting analyses. These results indicate that the dose dependent increase of glucocorticoid-inducible gene expression is caused by an increase in the number of transcriptionally active templates. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. Fig. 8. PMID:2167833

  6. Functional redundancy of two Pax-like proteins in transcriptional activation of cyst wall protein genes in Giardia lamblia.

    PubMed

    Chuang, Shen-Fung; Su, Li-Hsin; Cho, Chao-Cheng; Pan, Yu-Jiao; Sun, Chin-Hung

    2012-01-01

    The protozoan Giardia lamblia differentiates from a pathogenic trophozoite into an infectious cyst to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. Pax family transcription factors are involved in a variety of developmental processes in animals. Nine Pax proteins have been found to play an important role in tissue and organ development in humans. To understand the progression from primitive to more complex eukaryotic cells, we tried to identify putative pax genes in the G. lamblia genome and found two genes, pax1 and pax2, with limited similarity. We found that Pax1 may transactivate the encystation-induced cwp genes and interact with AT-rich initiatior elements that are essential for promoter activity and transcription start site selection. In this study, we further characterized Pax2 and found that, like Pax1, Pax2 was present in Giardia nuclei and it may specifically bind to the AT-rich initiator elements of the encystation-induced cwp1-3 and myb2 genes. Interestingly, overexpression of Pax2 increased the cwp1-3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of nuclear localization, DNA-binding activity, and transactivation activity of Pax2. These results are similar to those found in the previous Pax1 study. In addition, the profiles of gene expression in the Pax2 and Pax1 overexpressing cells significantly overlap in the same direction and ERK1 associated complexes may phosphorylate Pax2 and Pax1, suggesting that Pax2 and Pax1 may be downstream components of a MAPK/ERK1 signaling pathway. Our results reveal functional redundancy between Pax2 and Pax1 in up-regulation of the key encystation-induced genes. These results illustrate functional redundancy of a gene family can occur in order to increase maintenance of important gene function in the protozoan organism G. lamblia.

  7. Fluorescent activated cell sorting (FACS) combined with gene expression microarrays for transcription enrichment profiling of zebrafish lateral line cells.

    PubMed

    Gallardo, Viviana E; Behra, Martine

    2013-08-15

    Transgenic lines carrying fluorescent reporter genes like GFP have been of great value in the elucidation of developmental features and physiological processes in various animal models, including zebrafish. The lateral line (LL), which is a fish specific superficial sensory organ, is an emerging organ model for studying complex cellular processes in the context of the whole living animal. Cell migration, mechanosensory cell development/differentiation and regeneration are some examples. This sensory system is made of superficial and sparse small sensory patches called neuromasts, with less than 50 cells in any given patch. The paucity of cells is a real problem in any effort to characterize those cells at the transcriptional level. We describe here a method which we applied to efficiently separate subpopulation of cells of the LL, using two distinct stable transgenic zebrafish lines, Tg(cldnb:gfp) and Tg(tnks1bp1:EGFP). In both cases, the GFP positive (GFP+) cells were separated from the remainder of the animal by using a Fluorescent Activated Cell Sorter (FACS). The transcripts of the GFP+ cells were subsequently analyzed on gene expression microarrays. The combination of FACS and microarrays is an efficient method to establish a transcriptional signature for discrete cell populations which would otherwise be masked in whole animal preparation.

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

    PubMed

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

    2013-05-01

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

  9. Transcriptional Control of the TNF Gene

    PubMed Central

    Falvo, James V.; Tsytsykova, Alla V.; Goldfeld, Anne E.

    2016-01-01

    The cytokine TNF is a critical mediator of immune and inflammatory responses. The TNF gene is an immediate early gene, rapidly transcribed in a variety of cell types following exposure to a broad range of pathogens and signals of inflammation and stress. Regulation of TNF gene expression at the transcriptional level is cell type- and stimulus-specific, involving the recruitment of distinct sets of transcription factors to a compact and modular promoter region. In this review, we describe our current understanding of the mechanisms through which TNF transcription is specifically activated by a variety of extracellular stimuli in multiple cell types, including T cells, B cells, macrophages, mast cells, dendritic cells, and fibroblasts. We discuss the role of nuclear factor of activated T cells and other transcription factors and coactivators in enhanceosome formation, as well as the contradictory evidence for a role for nuclear factor κB as a classical activator of the TNF gene. We describe the impact of evolutionarily conserved cis-regulatory DNA motifs in the TNF locus upon TNF gene transcription, in contrast to the neutral effect of single nucleotide polymorphisms. We also assess the regulatory role of chromatin organization, epigenetic modifications, and long-range chromosomal interactions at the TNF locus. PMID:20173386

  10. Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

    PubMed

    Schmidt, R J; Ketudat, M; Aukerman, M J; Hoschek, G

    1992-06-01

    opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family.

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

    PubMed

    Groth, Rachel D; Mermelstein, Paul G

    2003-09-03

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

  12. The Fluoroquinolone Levofloxacin Triggers the Transcriptional Activation of Iron Transport Genes That Contribute to Cell Death in Streptococcus pneumoniae

    PubMed Central

    Ferrándiz, María-José

    2014-01-01

    We studied the transcriptomic response of Streptococcus pneumoniae to levofloxacin (LVX) under conditions inhibiting topoisomerase IV but not gyrase. Although a complex transcriptomic response was observed, the most outstanding result was the upregulation of the genes of the fatDCEB operon, involved in iron (Fe2+ and Fe3+) uptake, which were the only genes varying under every condition tested. Although the inhibition of topoisomerase IV by levofloxacin did not have a detectable effect in the level of global supercoiling, increases in general supercoiling and fatD transcription were observed after topoisomerase I inhibition, while the opposite was observed after gyrase inhibition with novobiocin. Since fatDCEB is located in a topological chromosomal domain downregulated by DNA relaxation, we studied the transcription of a copy of the 422-bp (including the Pfat promoter) region located upstream of fatDCEB fused to the cat reporter inserted into the chromosome 106 kb away from its native position: PfatfatD was upregulated in the presence of LVX in its native location, whereas no change was observed in the Pfatcat construction. Results suggest that topological changes are indeed involved in PfatfatDCE transcription. Upregulation of fatDCEB would lead to an increase of intracellular iron and, in turn, to the activation of the Fenton reaction and the increase of reactive oxygen species. In accordance, we observed an attenuation of levofloxacin lethality in iron-deficient media and in a strain lacking the gene coding for SpxB, the main source of hydrogen peroxide. In addition, we observed an increase of reactive oxygen species that contributed to levofloxacin lethality. PMID:24145547

  13. The fluoroquinolone levofloxacin triggers the transcriptional activation of iron transport genes that contribute to cell death in Streptococcus pneumoniae.

    PubMed

    Ferrándiz, María-José; de la Campa, Adela G

    2014-01-01

    We studied the transcriptomic response of Streptococcus pneumoniae to levofloxacin (LVX) under conditions inhibiting topoisomerase IV but not gyrase. Although a complex transcriptomic response was observed, the most outstanding result was the upregulation of the genes of the fatDCEB operon, involved in iron (Fe(2+) and Fe(3+)) uptake, which were the only genes varying under every condition tested. Although the inhibition of topoisomerase IV by levofloxacin did not have a detectable effect in the level of global supercoiling, increases in general supercoiling and fatD transcription were observed after topoisomerase I inhibition, while the opposite was observed after gyrase inhibition with novobiocin. Since fatDCEB is located in a topological chromosomal domain downregulated by DNA relaxation, we studied the transcription of a copy of the 422-bp (including the Pfat promoter) region located upstream of fatDCEB fused to the cat reporter inserted into the chromosome 106 kb away from its native position: PfatfatD was upregulated in the presence of LVX in its native location, whereas no change was observed in the Pfatcat construction. Results suggest that topological changes are indeed involved in PfatfatDCE transcription. Upregulation of fatDCEB would lead to an increase of intracellular iron and, in turn, to the activation of the Fenton reaction and the increase of reactive oxygen species. In accordance, we observed an attenuation of levofloxacin lethality in iron-deficient media and in a strain lacking the gene coding for SpxB, the main source of hydrogen peroxide. In addition, we observed an increase of reactive oxygen species that contributed to levofloxacin lethality.

  14. DNA methylation by CcrM activates the transcription of two genes required for the division of Caulobacter crescentus

    PubMed Central

    Gonzalez, Diego; Collier, Justine

    2013-01-01

    DNA methylation regulates many processes, including gene expression, by superimposing secondary information on DNA sequences. The conserved CcrM enzyme, which methylates adenines in GANTC sequences, is essential to the viability of several Alphaproteobacteria. In this study, we find that Caulobacter crescentus cells lacking the CcrM enzyme accumulate low levels of the two conserved FtsZ and MipZ proteins, leading to a severe defect in cell division. This defect can be compensated by the expression of the ftsZ gene from an inducible promoter or by spontaneous suppressor mutations that promote FtsZ accumulation. We show that CcrM promotes the transcription of the ftsZ and mipZ genes and that the ftsZ and mipZ promoter regions contain a conserved CGACTC motif that is critical to their activities and to their regulation by CcrM. In addition, our results suggest that the ftsZ promoter has the lowest activity when the CGACTC motif is non-methylated, an intermediate activity when it is hemi-methylated and the highest activity when it is fully methylated. The regulation of ftsZ expression by DNA methylation may explain why CcrM is essential in a subset of Alphaproteobacteria. PMID:23480529

  15. Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.

    PubMed

    Sakane, Yuto; Sakuma, Tetsushi; Kashiwagi, Keiko; Kashiwagi, Akihiko; Yamamoto, Takashi; Suzuki, Ken-Ichi T

    2014-01-01

    Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.

  16. Chromatin and DNA methylation dynamics during retinoic acid-induced RET gene transcriptional activation in neuroblastoma cells

    PubMed Central

    Angrisano, T.; Sacchetti, S.; Natale, F.; Cerrato, A.; Pero, R.; Keller, S.; Peluso, S.; Perillo, B.; Avvedimento, V. E.; Fusco, A.; Bruni, C. B.; Lembo, F.; Santoro, M.; Chiariotti, L.

    2011-01-01

    Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci. PMID:20952403

  17. Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins

    PubMed Central

    Jia, Jingyue; Bai, Fang; Jin, Yongxin; Santostefano, Katherine E.; Ha, Un-Hwan; Wu, Donghai

    2015-01-01

    The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies. Significance The present study describes a novel and powerful tool for the delivery of the genome editing enzyme transcription activator-like effector nuclease (TALEN) directly into pluripotent stem cells (PSCs), achieving desired base changes on the genomes of PSCs with high efficiency. This novel approach uses bacteria as a protein delivery

  18. Unique role of SRSF2 in transcription activation and diverse functions of the SR and hnRNP proteins in gene expression regulation.

    PubMed

    Mo, Sudong; Ji, Xiong; Fu, Xiang-Dong

    2013-01-01

    Transcription pause release from gene promoters has been recognized to be a critical point for transcriptional regulation in higher eukaryotes. Recent studies suggest that regulatory RNAs are extensively involved in transcriptional control, which may enlist various RNA binding proteins. We recently showed a key role of SRSF2, a member of the SR family of splicing regulators, in binding to promoter-associated small RNA to mediate transcription pause release, a regulatory strategy akin to the function of the HIV Tat protein via binding to the TAR element in nascent RNA to activate transcription. In this report, we further dissect the structural requirement for SRSF2 to function as a transcription activator and extend the analysis to multiple SR and hnRNP proteins by using the MS2 tethering strategy. Our results reveal that SRSF2 is a unique SR protein that activates transcription in a position-dependent manner while three other SR proteins enhance translation in a position-independent fashion. In contrast, multiple hnRNP proteins appear to negatively influence mRNA levels, especially when tethered in the gene body. These findings suggest broad participation of RNA binding proteins in diverse aspects of regulated gene expression at both the transcriptional and posttranscriptional levels in mammalian cells.

  19. Archaeal amoA and ureC genes and their transcriptional activity in the Arctic Ocean

    NASA Astrophysics Data System (ADS)

    Pedneault, Estelle; Galand, Pierre E.; Potvin, Marianne; Tremblay, Jean-Éric; Lovejoy, Connie

    2014-04-01

    Thaumarchaeota and the gene encoding for a subunit of ammonia monooxygenase (amoA) are ubiquitous in Polar Seas, and some Thaumarchaeota also have a gene coding for ureC, diagnostic for urease. Using quantitative PCR we investigated the occurrence of genes and transcripts of ureC and amoA in Arctic samples from winter, spring and summer. AmoA genes, ureC genes and amoA transcripts were always present, but ureC transcripts were rarely detected. Over a 48 h light manipulation experiment amoA transcripts persisted under light and dark conditions, but not ureC transcripts. In addition, maxima for amoA transcript were nearer the surface compared to amoA genes. Clone libraries using DNA template recovered shallow and deep amoA clades but only the shallow clade was recovered from cDNA (from RNA). These results imply environmental control of amoA expression with direct or indirect light effects, and rare ureC expression despite its widespread occurrence in the Arctic Ocean.

  20. Archaeal amoA and ureC genes and their transcriptional activity in the Arctic Ocean.

    PubMed

    Pedneault, Estelle; Galand, Pierre E; Potvin, Marianne; Tremblay, Jean-Éric; Lovejoy, Connie

    2014-04-11

    Thaumarchaeota and the gene encoding for a subunit of ammonia monooxygenase (amoA) are ubiquitous in Polar Seas, and some Thaumarchaeota also have a gene coding for ureC, diagnostic for urease. Using quantitative PCR we investigated the occurrence of genes and transcripts of ureC and amoA in Arctic samples from winter, spring and summer. AmoA genes, ureC genes and amoA transcripts were always present, but ureC transcripts were rarely detected. Over a 48 h light manipulation experiment amoA transcripts persisted under light and dark conditions, but not ureC transcripts. In addition, maxima for amoA transcript were nearer the surface compared to amoA genes. Clone libraries using DNA template recovered shallow and deep amoA clades but only the shallow clade was recovered from cDNA (from RNA). These results imply environmental control of amoA expression with direct or indirect light effects, and rare ureC expression despite its widespread occurrence in the Arctic Ocean.

  1. VID22 is required for transcriptional activation of the PSD2 gene in the yeast Saccharomyces cerevisiae.

    PubMed

    Miyata, Non; Miyoshi, Takuya; Yamaguchi, Takanori; Nakazono, Toshimitsu; Tani, Motohiro; Kuge, Osamu

    2015-12-15

    Phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae is synthesized through decarboxylation of phosphatidylserine (PS), catalysed by PS decarboxylase 1 (Psd1p) and 2 (Psd2p) and the cytidine 5'-diphosphate (CDP)-ethanolamine (CDP-Etn) pathway. PSD1 null (psd1Δ) and PSD2 null (psd2Δ) mutants are viable in a synthetic minimal medium, but a psd1Δ psd2Δ double mutant exhibits Etn auxotrophy, which is incorporated into PE through the CDP-Etn pathway. We have previously shown that psd1Δ is synthetic lethal with deletion of VID22 (vid22Δ) [Kuroda et al. (2011) Mol. Microbiol. 80: , 248-265]. In the present study, we found that vid22Δ mutant exhibits Etn auxotrophy under PSD1-depressed conditions. Deletion of VID22 in wild-type and PSD1-depressed cells caused partial defects in PE formation through decarboxylation of PS. The enzyme activity of PS decarboxylase in an extract of vid22Δ cells was ∼70% of that in wild-type cells and similar to that in psd2Δ cells and the PS decarboxylase activity remaining in the PSD1-depressed cells became almost negligible with deletion of VID22. Thus, the vid22Δ mutation was suggested to cause a defect in the Psd2p activity. Furthermore, vid22Δ cells were shown to be defective in expression of the PSD2 gene tagged with 6×HA, the defect being ameliorated by replacement of the native promoter of the PSD2 gene with a CYC1 promoter. In addition, an α-galactosidase reporter assay revealed that the activity of the promoter of the PSD2 gene in vid22Δ cells was ∼5% of that in wild-type cells. These results showed that VID22 is required for transcriptional activation of the PSD2 gene.

  2. Sublethal effects of the flame retardant intermediate hexachlorocyclopentadiene (HCCPD) on the gene transcription and protein activity of Daphnia magna.

    PubMed

    Houde, Magali; Carter, Barbara; Douville, Mélanie

    2013-09-15

    Hexachlorocyclopentadiene (HCCPD) is a chlorinated chemical of high production volume used as an intermediate in the production of flame retardants. HCCPD may be released to the environment during production, use, and as a result of product degradation. The objectives of this study were to evaluate sublethal effects of HCCPD exposure to Daphnia magna at environmentally relevant concentrations (0.0138-13.8 μg/L) using genomic tools (microarray and qPCR), enzyme activities, and life-history endpoints (survival, reproduction, and growth). In chronic exposures, no differences were observed in life-history endpoints (survival, time of first brood, time of first molt, molt frequency, number of neonates, and body length) between exposed organisms and controls. Microarray analyses indicated significant differential genomic transcription for 46 genes (p-value ≤ 0.05 and fold-change>2). Five identified genes were related to metabolic functions. Enzyme activities of α-amylase and trypsin, selected based on transcriptional responses, were evaluated in D. magna. Although trypsin activity was similar between treatments and controls, the activity of α-amylase significantly decreased with increasing HCCPD concentrations. On the chemical level, instability of HCCPD was observed in spiked culture media, most probably due to photolysis and biodegradation. HCCPD was not detected in surface water samples collected upstream and at the point of discharge of a major wastewater treatment plant effluent. Environmentally, rapid degradation of HCCPD could be outdone by its continuous release into aquatic ecosystems in specific areas of concern (e.g., vicinity of industries and hazardous sites). Toxicity results from this study highlight the use of genomics in the identification of biomarkers and help advance the science, and potential use, of multi-level biological approaches for environmental risk assessment.

  3. Linking Smads and transcriptional activation.

    PubMed

    Inman, Gareth J

    2005-02-15

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

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

    PubMed

    Lainšček, Duško; Lebar, Tina; Jerala, Roman

    2017-02-26

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

  5. Fli-1 controls transcription from the MCP-1 gene promoter, which may provide a novel mechanism for chemokine and cytokine activation.

    PubMed

    Lennard Richard, Mara L; Nowling, Tamara K; Brandon, Danielle; Watson, Dennis K; Zhang, Xian K

    2015-02-01

    Regulation of proinflammatory cytokines and chemokines is a primary role of the innate immune response. MCP-1 is a chemokine that recruits immune cells to sites of inflammation. Expression of MCP-1 is reduced in primary kidney endothelial cells from mice with a heterozygous knockout of the Fli-1 transcription factor. Fli-1 is a member of the Ets family of transcription factors, which are evolutionarily conserved across several organisms including Drosophilla, Xenopus, mouse and human. Ets family members bind DNA through a consensus sequence GGAA/T, or Ets binding site (EBS). Fli-1 binds to EBSs within the endogenous MCP-1 promoter by ChIP assay. In this study, transient transfection assays indicate that the Fli-1 gene actively promotes transcription from the MCP-1 gene promoter in a dose-dependent manner. Mutation of the DNA binding domain of Fli-1 demonstrated that Fli-1 activates transcription of MCP-1 both directly, by binding to the promoter, and indirectly, likely through interactions with other transcription factors. Another Ets transcription factor, Ets-1, was also tested, but failed to promote transcription. While Ets-1 failed to drive transcription independently, a weak synergistic activation of the MCP-1 promoter was observed between Ets-1 and Fli-1. In addition, Fli-1 and the NFκB family member p65 were found to interact synergistically to activate transcription from the MCP-1 promoter, while Sp1 and p50 inhibit this interaction. Deletion studies identified that EBSs in the distal and proximal MCP-1 promoter are critical for Fli-1 activation from the MCP-1 promoter. Together, these results demonstrate that Fli-1 is a novel regulator of the proinflammatory chemokine MCP-1, that interacts with other transcription factors to form a complex transcriptional mechanism for the activation of MCP-1 and mediation of the inflammatory response.

  6. MALT1 auto-proteolysis is essential for NF-κB-dependent gene transcription in activated lymphocytes.

    PubMed

    Baens, Mathijs; Bonsignore, Luca; Somers, Riet; Vanderheydt, Charlotte; Weeks, Stephen D; Gunnarsson, Jenny; Nilsson, Ewa; Roth, Robert G; Thome, Margot; Marynen, Peter

    2014-01-01

    Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.

  7. Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes.

    PubMed

    Li, Chunliang; Qi, Rong; Singleterry, Rebecca; Hyle, Judith; Balch, Amanda; Li, Xiuling; Sublett, Jack; Berns, Hartmut; Valentine, Marcus; Valentine, Virginia; Sherr, Charles J

    2014-05-01

    Injection of transcription activator-like effector nucleases (TALEN) mRNAs into mouse zygotes transferred into foster mothers efficiently generated founder mice with heritable mutations in targeted genes. Immunofluorescence visualization of phosphorylated histone 2A (γH2AX) combined with fluorescence in situ hybridization revealed that TALEN pairs targeting the Agouti locus induced site-directed DNA breaks in zygotes within 6 h of injection, an activity that continued at reduced efficiency in two-cell embryos. TALEN-Agouti mRNAs injected into zygotes of brown FvB × C57BL/6 hybrid mice generated completely black pups, confirming that mutations were induced prior to, and/or early after, cell division. Founder mice, many of which were mosaic, transmitted altered Agouti alleles to F1 pups to yield an allelic series of mutant strains. Although mutations were targeted to "spacer" sequences flanked by TALEN binding sites, larger deletions that extended beyond the TALEN-binding sequences were also detected and were similarly inherited through the germ line. Zygotic coinjection of TALEN mRNAs directed to the Agouti, miR-205, and the Arf tumor suppressor loci yielded pups containing frequent and heritable mutations of two or three genes. Simultaneous gene editing in zygotes affords an efficient approach for producing mice with compound mutant phenotypes, bypassing constraints of conventional mouse knockout technology in embryonic stem cells.

  8. Targeted Ablation Reveals a Novel Role of FKBP52 in Gene-Specific Regulation of Glucocorticoid Receptor Transcriptional Activity

    PubMed Central

    Wolf, Irene M.; Periyasamy, Sumudra; Hinds, Terry; Yong, Weidong; Shou, Weinian; Sanchez, Edwin R.

    2009-01-01

    FKBP52 is a tetratricopeptide repeat (TPR) protein with peptidyl-prolyl isomerase activity and is found in steroid receptor complexes, including glucocorticoid receptor (GR). It is generally accepted that FKBP52 has a stimulatory effect on GR transcriptional activity. However, the mechanism by which FKBP52 controls GR is not yet clear, with reports showing effects on GR hormone-binding affinity and/or hormone-induced nuclear translocation. To address this issue, we have generated mice with targeted ablation of the FKBP52 gene. To date, no overt defects of GR-regulated physiology have been found in these animals, demonstrating that FKBP52 is not an essential regulator of global GR activity. To better assess the impact of FKBP52 on GR, mouse embryonic fibroblasts (MEFs) were generated from wild-type (WT) and FKBP52-deficient (KO) animals. Analysis of GR activity at reporter genes showed an approximate 70% reduction of activity in 52KO MEF cells, with no effect of FKBP52 loss on thyroid receptor. Interestingly, GR activity at endogenous genes was not globally affected in 52KO cells, with reduced activity at GILZ and FKBP51, but not at SGK and p21. Thus, FKBP52 appears to be a gene-specific modulator of GR. To investigate the mechanism of this action, analyses of GR heterocomplex composition, hormone-binding affinity, and ability to undergo hormone-induced nuclear translocation and DNA-binding were performed. Interestingly, no effect of FKBP52 loss was found for any of these GR properties, suggesting that the main function of FKBP52 is a heretofore-unknown ability to control GR activity at target genes. Lastly, loss of FKBP52 did not affect the ability of GR to undergo hormone-induced autologous down-regulation, showing that FKBP52 does not contribute to all branches of GR signaling. The implications of these results to the potential actions of FKBP52 on GR activity in vivo are discussed. PMID:19073255

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

    PubMed Central

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

    2013-01-01

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

  10. Transcriptional activation of RNA polymerase III-dependent genes by the human T-cell leukemia virus type 1 tax protein.

    PubMed Central

    Gottesfeld, J M; Johnson, D L; Nyborg, J K

    1996-01-01

    The human T-cell leukemia virus-encoded tax protein is a potent activator of many viral and cellular genes transcribed by RNA polymerase II. We find that both chromatin and cell extracts derived from human T-cell leukemia virus type 1-infected human T lymphocytes support higher levels of 5S rRNA and tRNA gene transcription than chromatin or extracts from uninfected T lymphocytes. The viral protein Tax was likely responsible for this higher level of class II gene transcription, as purified Tax was found to stimulate both genes when added to the uninfected cell extract or in reconstituted systems. Both limiting-component transcription assays and DNA binding assays identified the class III gene transcription factor TFIIIB as the principle target of Tax activity. Surprisingly, we find that Tax increases the effective concentration of active TFIIIB molecules. These data suggest that Tax stimulates RNA polymerase III-dependent gene expression by accelerating the rate and/or extent of transcription initiation complex assembly. PMID:8657153

  11. The catabolite gene activator protein (CAP) is not required for indole-3-acetic acid to activate transcription of the araBAD operon of Escherichia coli K-12.

    PubMed

    Ebright, R H; Beckwith, J

    1985-01-01

    Kline et al. (1980) have reported that indole-3-acetic acid (IAA) and four other indole derivatives are able to substitute for cAMP in activating expression of the ara regulon of E. coli. We have examined this phenomenon in detail, utilizing fusions between the structural gene for beta-galactosidase and the promoters for the araBAD, araE, and araFG operons. We confirm that IAA potently stimulates transcription from the araBAD promoter. The effect is highly specific to araBAD, as IAA has no, or only slight, effects on the araE and araFG operons. However, contrary to the results of Kline et al., we find that the action of IAA does not require CAP. Thus, IAA fully stimulates the transcription of araBAD in a strain which bears a complete deletion of the crp gene.

  12. Core promoter-specific gene regulation: TATA box selectivity and Initiator-dependent bi-directionality of serum response factor-activated transcription.

    PubMed

    Xu, Muyu; Gonzalez-Hurtado, Elsie; Martinez, Ernest

    2016-04-01

    Gene-specific activation by enhancers involves their communication with the basal RNA polymerase II transcription machinery at the core promoter. Core promoters are diverse and may contain a variety of sequence elements such as the TATA box, the Initiator (INR), and the downstream promoter element (DPE) recognized, respectively, by the TATA-binding protein (TBP) and TBP-associated factors of the TFIID complex. Core promoter elements contribute to the gene selectivity of enhancers, and INR/DPE-specific enhancers and activators have been identified. Here, we identify a TATA box-selective activating sequence upstream of the human β-actin (ACTB) gene that mediates serum response factor (SRF)-induced transcription from TATA-dependent but not INR-dependent promoters and requires the TATA-binding/bending activity of TBP, which is otherwise dispensable for transcription from a TATA-less promoter. The SRF-dependent ACTB sequence is stereospecific on TATA promoters but activates in an orientation-independent manner a composite TATA/INR-containing promoter. More generally, we show that SRF-regulated genes of the actin/cytoskeleton/contractile family tend to have a TATA box. These results suggest distinct TATA-dependent and INR-dependent mechanisms of TFIID-mediated transcription in mammalian cells that are compatible with only certain stereospecific combinations of activators, and that a TBP-TATA binding mechanism is important for SRF activation of the actin/cytoskeleton-related gene family.

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

    PubMed

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

    2015-11-05

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

  14. Cell Type-Specific and Inducible PTEN Gene Silencing by a Tetracycline Transcriptional Activator-Regulated Short Hairpin RNA.

    PubMed

    Wang, Shan; Wang, Ting; Wang, Tao; Jia, Lintao

    2015-11-01

    Inducible and reversible gene silencing in desired types of cells is instrumental for deciphering gene functions using cultured cells or in vivo models. However, efficient conditional gene knockdown systems remain to be established. Here, we report the generation of an inducible expression system for short hairpin RNA (shRNA) targeted to PTEN, a well-documented dual-specificity phosphatase involved in tumor suppression and ontogenesis. Upon induction by doxycycline (DOX), the reverse tetracycline transcriptional activator (rtTA) switched on the concomitant expression of GFP and a miR-30 precursor, the subsequent processing of which released the embedded PTEN-targeted shRNA. The efficacy and reversibility of PTEN knockdown by this construct was validated in normal and neoplastic cells, in which PTEN deficiency resulted in accelerated cell proliferation, suppressed apoptosis, and increased invasiveness. Transgenic mice harboring the conditional shRNA-expression cassette were obtained; GFP expression and concurrent PTEN silencing were observed upon ectopic expression of rtTA and induction with Dox. Therefore, this study provides novel tools for the precise dissection of PTEN functions and the generation of PTEN loss of function models in specific subsets of cells during carcinogenesis and ontogenesis.

  15. Genetic diversity of transcriptional activator-like effector genes in Chinese isolates of Xanthomonas oryzae pv. oryzicola.

    PubMed

    Ji, Zhi-Yuan; Zakria, Muhammad; Zou, Li-Fang; Xiong, Li; Li, Zheng; Ji, Guang-Hai; Chen, Gong-You

    2014-07-01

    Xanthomonas oryzae pv. oryzicola causes bacterial leaf streak (BLS), a devastating disease of rice in Asia countries. X. oryzae pv. oryzicola utilizes repertoires of transcriptional activator-like effectors (TALEs) to manipulate host resistance or susceptibility; thus, TALEs can determine the outcome of BLS. In this report, we studied genetic diversity in putative tale genes of 65 X. oryzae pv. oryzicola strains that originated from nine provinces of southern China. Genomic DNAs from the 65 strains were digested with BamHI and hybridized with an internal fragment of avrXa3, a tale gene originating from the related pathogen, X. oryzae pv. oryzae, which causes bacterial leaf blight (BLB). Southern blot analysis indicated that the strains contained a variable number (9 to 22) of avrXa3-hybridizing fragments (e.g., putative tale genes). Based on the number and size of hybridizing bands, strains were classified into 14 genotypes (designated 1 to 14), and genotypes 3 and 10 represented 29.23 and 24.64% of the total, respectively. A high molecular weight BamHI fragment (HMWB; ≈6.0 kb) was present in 12 of the 14 genotypes, and sequence analysis of the HMWB revealed the presence of a C-terminally truncated tale, an insertion element related to IS1403, and genes encoding phosphoglycerate mutase and endonuclease V. Primers were developed from the 6.0-kb HMWB fragment and showed potential in genotyping X. oryzae pv. oryzicola strains by polymerase chain reaction. Virulence of X. oryzae pv. oryzicola strains was assessed on 23 rice cultivars containing different resistance genes for BLB. The X. oryzae pv. oryzicola strains could be grouped into 14 pathotypes (I to XIV), and the grouping of strains was almost identical to the categories determined by genotypic analysis. In general, strains containing higher numbers of putative tale genes were more virulent on rice than strains containing fewer tales. The results also indicate that there are no gene-for-gene relationships

  16. Body-hypomethylated human genes harbor extensive intragenic transcriptional activity and are prone to cancer-associated dysregulation.

    PubMed

    Mendizabal, Isabel; Zeng, Jia; Keller, Thomas E; Yi, Soojin V

    2017-01-23

    Genomic DNA methylation maps (methylomes) encode genetic and environmental effects as stable chemical modifications of DNA. Variations in DNA methylation, especially in regulatory regions such as promoters and enhancers, are known to affect numerous downstream processes. In contrast, most transcription units (gene bodies) in the human genome are thought to be heavily methylated. However, epigenetic reprogramming in cancer often involves gene body hypomethylation with consequences on gene expression. In this study, we focus on the relatively unexplored phenomenon that some gene bodies are devoid of DNA methylation under normal conditions. Utilizing nucleotide-resolution methylomes of diverse samples, we show that nearly 2000 human genes are commonly hypomethylated. Remarkably, these genes occupy highly specialized genomic, epigenomic, evolutionary and functional niches in our genomes. For example, hypomethylated genes tend to be short yet encode significantly more transcripts than expected based upon their lengths, include many genes involved in nucleosome and chromatin formation, and are extensively and significantly enriched for histone-tail modifications and transcription factor binding with particular relevance for cis-regulation. Furthermore, they are significantly more prone to cancer-associated hypomethylation and mutation. Consequently, gene body hypomethylation represents an additional layer of epigenetic regulatory complexity, with implications on cancer-associated epigenetic reprogramming.

  17. Allele-specific transcriptional activity of the variable number of tandem repeats in 5' region of the DRD4 gene is stimulus specific in human neuronal cells.

    PubMed

    Paredes, U M; Quinn, J P; D'Souza, U M

    2013-03-01

    The dopamine receptor D4 (DRD4) gene includes several variable number of tandem repeat loci that have been suggested to modulate DRD4 gene expression patterns. Previous studies showed differential basal activity of the two most common variants of a tandem repeat (120 bp per repeat unit) located in the 5' region adjacent to the DRD4 promoter in human cell lines. In this communication, we further characterized the ability of this polymorphic repeat to elicit tissue-, allele- and stimuli-specific transcriptional activity in vitro. The short and long variants of the DRD4 5' tandem repeat were cloned into a luciferase reporter gene construct containing the SV40 promoter. The luciferase constructs were cotransfected with expression vectors of two ubiquitously expressed human transcription factors (TFs), CCCTC-binding factor (CTCF) and upstream stimulatory factor 2 (USF2), into human cell lines and primary cultures of neonate rat cortex and luciferase activity measured. Overexpression with these TFs resulted in differential cell- and allele-specific transcriptional activities of the luciferase constructs. The results of our experiments show that variants of this tandem repeat in the 5' promoter of the DRD4 gene will direct differential reporter gene transcriptional activity in a cell-type-specific manner dependent on the signal pathways activated.

  18. Transcript Analysis of White spot syndrome virus Latency and Phagocytosis Activating Protein Genes in Infected Shrimp (Penaeus monodon).

    PubMed

    Shekhar, M S; Dillikumar, M; Vinaya Kumar, K; Gopikrishna, G; Rajesh, S; Kiruthika, J; Ponniah, A G

    2012-12-01

    Viral latency has been recently observed to be associated with White spot syndrome virus (WSSV) infection in shrimp. In the present study, shrimp samples (Penaeus monodon) surviving WSSV infection were examined for presence of WSSV in latent phase. Virus latency was observed in shrimp which were either experimentally challenged with WSSV and survived the infection or those which survived the natural infection. Three viral transcripts (ORFs 427, 151, 366) associated with latency were analyzed by real-time PCR. The shrimp surviving the natural WSSV infection on estimation with RT-PCR were found to have low grade of WSSV infection (less than 56 copies of WSSV). All the shrimp samples were RT-PCR negative for structural protein genes of WSSV, VP24 and VP28, indicating that these samples were harboring latent phase virus. RT-PCR of all the shrimp samples which survived WSSV infection revealed amplification of phagocytosis activating protein (PAP) gene (435 bp) with higher gene expression levels in experimentally challenged shrimp when compared to naturally infected shrimp. The expression of PAP in WSSV infected shrimp samples indicates its possible role in host response for resistance against WSSV infection. PAP was cloned and expressed as recombinant protein for protection studies. Shrimp were injected with three doses (5, 15 and 20 μg g(-1) body weight) of recombinant PAP. Relative percent survival of 10 % was observed in shrimp immunized with the dose of 15 μg g(-1) body weight of recombinant PAP. The expression of both WSSV latency associated and PAP genes obtained from shrimp surviving the WSSV infection, indicates the possible role of these genes in host-pathogen interaction.

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

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

    PubMed

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

    2014-12-01

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

  1. Phorate-induced oxidative stress, DNA damage and transcriptional activation of p53 and caspase genes in male Wistar rats

    SciTech Connect

    Saquib, Quaiser; Attia, Sabry M.; Siddiqui, Maqsood A.; Aboul-Soud, Mourad A.M.; Al-Khedhairy, Abdulaziz A.; Giesy, John P.; Musarrat, Javed

    2012-02-15

    Male Wistar rats exposed to a systemic organophosphorus insecticide, phorate [O,O-diethyl S-[(ethylthio) methyl] phosphorothioate] at varying oral doses of 0.046, 0.092 or 0.184 mg phorate/kg bw for 14 days, exhibited substantial oxidative stress, cellular DNA damage and activation of apoptosis-related p53, caspase 3 and 9 genes. The histopathological changes including the pyknotic nuclei, inflammatory leukocyte infiltrations, renal necrosis, and cardiac myofiber degeneration were observed in the liver, kidney and heart tissues. Biochemical analysis of catalase and glutathione revealed significantly lesser activities of antioxidative enzymes and lipid peroxidation in tissues of phorate exposed rats. Furthermore, generation of intracellular reactive oxygen species and reduced mitochondrial membrane potential in bone marrow cells confirmed phorate-induced oxidative stress. Significant DNA damage was measured through comet assay in terms of the Olive tail moment in bone marrow cells of treated animals as compared to control. Cell cycle analysis also demonstrated the G{sub 2}/M arrest and appearance of a distinctive SubG{sub 1} peak, which signified induction of apoptosis. Up-regulation of tumor suppressor p53 and caspase 3 and 9 genes, determined by quantitative real-time PCR and enzyme-linked immunosorbent assay, elucidated the activation of intrinsic apoptotic pathways in response to cellular stress. Overall, the results suggest that phorate induces genetic alterations and cellular toxicity, which can adversely affect the normal cellular functioning in rats. -- Highlights: ► This is the first report on molecular toxicity of phorate in an in vivo test system. ► Phorate induces biochemical and histological changes in liver, kidney and heart. ► Rats treated with phorate exhibited DNA damage in bone marrow cells. ► Phorate induces apoptosis, oxidative stress and alters mitochondrial fluorescence. ► Phorate induces transcriptional changes and enhanced

  2. Resveratrol post-transcriptionally regulates pro-inflammatory gene expression via regulation of KSRP RNA binding activity

    PubMed Central

    Bollmann, Franziska; Art, Julia; Henke, Jenny; Schrick, Katharina; Besche, Verena; Bros, Matthias; Li, Huige; Siuda, Daniel; Handler, Norbert; Bauer, Florian; Erker, Thomas; Behnke, Felix; Mönch, Bettina; Härdle, Lorena; Hoffmann, Markus; Chen, Ching-Yi; Förstermann, Ulrich; Dirsch, Verena M.; Werz, Oliver; Kleinert, Hartmut; Pautz, Andrea

    2014-01-01

    Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol. PMID:25352548

  3. Stress responses in alfalfa (Medicago sativa L.) XIX. Transcriptional activation of oxidative pentose phosphate pathway genes at the onset of the isoflavonoid phytoalexin response.

    PubMed

    Fahrendorf, T; Ni, W; Shorrosh, B S; Dixon, R A

    1995-08-01

    We have isolated cDNA clones encoding the pentose phosphate pathway enzymes 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from alfalfa (Medicago sativa L.). These exhibit extensive nucleotide and amino acid sequence similarity to the corresponding genes from bacteria, Drosophila and mammals. Transcripts encoding both enzymes are expressed at high levels in roots and nodules. Exposure of alfalfa suspension cells to an elicitor from yeast cell walls results in co-ordinated increases in transcription rates for both genes, followed by increased steady state transcript levels but only slightly increased extractable enzyme activities, at the onset of accumulation of isoflavonoid phytoalexins. Levels of NADPH and NADP remain relatively constant in alfalfa cells following elicitation. The rapid transcriptional activation of 6PGDH and G6PDH does not therefore appear to be a response to altered pyridine nucleotide redox state. These genes appear to respond to early events in elicitor-mediated signalling rather than to subsequent elicitor-induced changes in secondary metabolism. Hydrogen peroxide, a potential signal for elicitation of anti-oxidative genes in biologically stressed plant cells, did not induce 6PGDH or G6PDH transcripts or enzymatic activity.

  4. Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

    PubMed

    Weyman, Philip D; Beeri, Karen; Lefebvre, Stephane C; Rivera, Josefa; McCarthy, James K; Heuberger, Adam L; Peers, Graham; Allen, Andrew E; Dupont, Christopher L

    2015-05-01

    Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.

  5. Site-specific gene targeting using transcription activator-like effector (TALE)-based nuclease in Brassica oleracea.

    PubMed

    Sun, Zijian; Li, Nianzu; Huang, Guodong; Xu, Junqiang; Pan, Yu; Wang, Zhimin; Tang, Qinglin; Song, Ming; Wang, Xiaojia

    2013-11-01

    Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases (TALENs), constructed using a method called "unit assembly", specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non-homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.

  6. An initiation site of DNA replication with transcriptional enhancer activity present upstream of the c-myc gene.

    PubMed Central

    Iguchi-Ariga, S M; Okazaki, T; Itani, T; Ogata, M; Sato, Y; Ariga, H

    1988-01-01

    We have previously reported that c-myc protein may promote cellular DNA replication by binding to initiation sites of replication. Here we report that a putative origin of human cellular DNA replication (ori) is present at approximately 2 kb upstream of the coding region of the c-myc gene itself. The c-myc protein, or protein(s) complexed with c-myc protein, bind to the upstream region (approximately 200 bp in length) which has transcriptional enhancer activity as well as autonomously replicating activity in human cells, suggesting that the c-myc protein may be an enhancer binding protein as well as a DNA replication protein. Results with deletion mutants suggest that the sequence essential to the origin of DNA replication may be adjacent to, but cannot be clearly separated from, the sequence responsible for enhancer activity. Furthermore, when cloned DNA containing putative c-myc protein binding sequences was transfected as competitor into HL-60 cells, expression of c-myc was inhibited, suggesting that c-myc protein itself may be necessary for c-myc expression. Images PMID:3053161

  7. Transcriptional activation of the Azotobacter vinelandii polyhydroxybutyrate biosynthetic genes phbBAC by PhbR and RpoS.

    PubMed

    Hernandez-Eligio, Alberto; Castellanos, Mildred; Moreno, Soledad; Espín, Guadalupe

    2011-11-01

    We previously showed that in Azotobacter vinelandii, accumulation of polyhydroxybutyrate (PHB) occurs mainly during the stationary phase, and that a mutation in phbR, encoding a transcriptional regulator of the AraC family, reduces PHB accumulation. In this study, we characterized the roles of PhbR and RpoS, a central regulator during stationary phase in bacteria, in the regulation of expression of the PHB biosynthetic operon phbBAC and phbR. We showed that inactivation of rpoS reduced PHB accumulation, similar to the phbR mutation, and inactivation of both rpoS and phbR resulted in an inability to produce PHB. We carried out expression studies with the wild-type, and the rpoS, phbR and double rpoS-phbR mutant strains, using quantitative RT-PCR, as well as phbB : : gusA and phbR : : gusA gene fusions. These studies showed that both PhbR and RpoS act as activators of phbB and phbR, and revealed a role for PhbR as an autoactivator. We also demonstrated that PhbR binds specifically to two almost identical 18 bp sites, TGTCACCAA-N(4)-CACTA and TGTCACCAA-N(4)-CAGTA, present in the phbB promoter region. The activation of phbB and phbR transcription by RpoS reported here is in agreement with the observation that accumulation of PHB in A. vinelandii occurs mainly during the stationary phase.

  8. Transcriptional effects of gene dose reduction

    PubMed Central

    2014-01-01

    Large-scale gene dose reductions usually lead to abnormal phenotypes or death. However, male mammals, Drosophila, and Caenorhabditis elegans have only one X chromosome and thus can be considered as monosomic for a major chromosome. Despite the deleterious effects brought about by such gene dose reduction in the case of an autosome, X chromosome monosomy in males is natural and innocuous. This is because of the nearly full transcriptional compensation for X chromosome genes in males, as opposed to no or partial transcriptional compensation for autosomal one-dose genes arising due to deletions. Buffering, the passive absorption of disturbance due to enzyme kinetics, and feedback responses triggered by expression change contribute to partial compensation. Feed-forward mechanisms, which are active responses to genes being located on the X, rather than actual gene dose are important contributors to full X chromosome compensation. In the last decade, high-throughput techniques have provided us with the tools to effectively and quantitatively measure the small-fold transcriptional effects of dose reduction. This is leading to a better understanding of compensatory mechanisms. PMID:24581086

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

    PubMed Central

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

    2007-01-01

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

  10. Involvement of NF-Y and Sp1 in basal and cAMP-stimulated transcriptional activation of the tryptophan hydroxylase (TPH ) gene in the pineal gland.

    PubMed

    Côté, F; Schussler, N; Boularand, S; Peirotes, A; Thévenot, E; Mallet, J; Vodjdani, G

    2002-05-01

    The expression of the tryptophan hydroxylase (TPH) gene, encoding the rate-limiting enzyme of serotonin biosynthesis, is tightly regulated both at the transcriptional and at the post-transcriptional levels. In the pineal gland, transcription of the gene is activated in response to an intracellular circadian increase of the cAMP concentration. We have previously shown that transcription of a 2.1-kb fragment of the human TPH promoter is induced by cAMP, although it lacks the canonical cAMP responsive element, CRE. The minimal promoter (-73/+29) has only weak transcriptional activity but is responsive to cAMP. It contains an inverted CCAAT box, which was demonstrated to be involved in this response. Here, we have extended our investigation to the functional features of the inverted CCAAT box in the -252/+29 TPH promoter, which has a higher basal activity. We show that an additional cis -acting sequence, the adjacent GC-rich region, cooperates with the inverted CCAAT box for the full activation of basal transcription, and that both elements are essential for the full cAMP response. We also show that in pinealocytes, NF-Y and Sp1 transactivators bind the inverted CCAAT box and GC-rich-region, respectively. These factors participate in a novel pathway for the cAMP-mediated response of the TPH promoter, which is independent of the canonical CRE-mediated response.

  11. Activity (transcription) of the genes for MLH1, MSH2 and p53 in sporadic colorectal tumours with micro-satellite instability.

    PubMed

    Tou, S I H; Drye, E R; Boulos, P B; Hollingsworth, S J

    2004-05-17

    Micro-satellite instability (MSI) is relevant in the management of colorectal cancers (CRC) and relies on analysis of gene mutations, or production of the proteins involved in DNA mismatch repair (e.g. MLH1, MSH2). p53 mutation is also relevant in MSI, but high-level CRC (MSI-H) demonstrate fewer mutations than low-level (MSI-L) or stable (MSS) cancers. Recently, the importance of gene activity (transcription) in MSI has been identified, where rather than being mutated genes have been downregulated. In this study, 67 sporadic CRC and eight samples of normal bowel were analysed for MSI status (by SSCP) and levels of MLH1, MSH2 and p53 gene transcription (by RT-PCR and scanning densitometry). Micro-satellite instability correlated with gender and site, with more MSI-H CRC in females (P<0.02) and in the right colon (P<0.04). In MSI-H, p53 transcription was markedly reduced (P<0.003). Compared to normal bowel, MLH1 transcription was elevated in all cancers (P<0.01), while MSH2 transcription was elevated only in MSI-H (P<0.04). There was a direct correlation between MLH1 and MSH2 transcription (P<0.001). Although fewer mutations are reported in MSI-H than MSI-L/MSS, these results suggest that reduced p53 transcription might account for decreased tumour suppression in MSI-H. The direct correlation between MLH1 and MSH2 transcription suggests that control of these genes might be coordinated.

  12. Transcription of the Salmonella Invasion Gene Activator, hilA, Requires HilD Activation in the Absence of Negative Regulators

    PubMed Central

    Boddicker, Jennifer D.; Knosp, Boyd M.; Jones, Bradley D.

    2003-01-01

    Salmonella enterica serovar Typhimurium causes human gastroenteritis and a systemic typhoid-like infection in mice. Infection is initiated by entry of the bacteria into intestinal epithelial cells and is mediated by a type III secretion system that is encoded by genes in Salmonella pathogenicity island 1. The expression of invasion genes is tightly regulated by environmental conditions such as oxygen and osmolarity, as well as by many bacterial factors. The hilA gene encodes an OmpR/ToxR family transcriptional regulator that activates the expression of invasion genes in response to both environmental and genetic regulatory factors. HilD is an AraC/XylS regulator that has been postulated to act as a derepressor of hilA expression that promotes transcription by interfering with repressor binding at the hilA promoter. Our research group has identified four genes (hilE, hha, pag, and ams) that negatively affect hilA transcription. Since the postulated function of HilD at the hilA promoter is to counteract the effects of repressors, we examined this model by measuring hilA::Tn5lacZY expression in strains containing negative regulator mutations in the presence or absence of functional HilD. Single negative regulator mutations caused significant derepression of hilA expression, and two or more negative regulator mutations led to very high level expression of hilA. However, in all strains tested, the absence of hilD resulted in low-level expression of hilA, suggesting that HilD is required for activation of hilA expression, whether or not negative regulators are present. We also observed that deletion of the HilD binding sites in the chromosomal hilA promoter severely decreased hilA expression. In addition, we found that a single point mutation at leucine 289 in the C-terminal domain of the α subunit of RNA polymerase leads to very low levels of hilA::Tn5lacZY expression, suggesting that HilD activates transcription of hilA by contacting and recruiting RNA polymerase to

  13. Nrf2 Transcription Factor Can Directly Regulate mTOR: LINKING CYTOPROTECTIVE GENE EXPRESSION TO A MAJOR METABOLIC REGULATOR THAT GENERATES REDOX ACTIVITY.

    PubMed

    Bendavit, Gabriel; Aboulkassim, Tahar; Hilmi, Khalid; Shah, Sujay; Batist, Gerald

    2016-12-02

    Nrf2 is a master transcription factor that regulates a wide variety of cellular proteins by recognizing and binding to antioxidant response elements (AREs) in their gene promoter regions. In this study we show that increasing cellular Nrf2 results in transcriptional activation of the gene for mTOR, which is central to the PI3K signaling pathway. This is the case in cells with normal physiological PI3K. However, in cells with abnormally active PI3K increased cellular Nrf2 levels have no effect on mTOR. ChIP assays results show that increased Nrf2 binding is associated with decreased p65 binding and H3-K27me3 signal (marker of gene repression) as well as increased H3-K4me3 signal (marker of gene activation). However, in cells with PI3K activation, no effect of cellular Nrf2 increase on mTOR transcription was observed. In these cells, increasing Nrf2 levels increases Nrf2 promoter binding marginally, whereas p65 binding and H3-K27me3 mark were significantly increased, and H3-K4me3 signal is reduced. Together, these data show for the first time that Nrf2 directly regulates mTOR transcription when the PI3K pathway is intact, whereas this function is lost when PI3K is activated. We have identified a link between the Nrf2 system of sensing environmental stress and mTOR, which is a key cellular protein in metabolism. Studies in cells with activating mutations in the PI3K pathway suggest that Nrf2 transcriptional regulation of mTOR is related to promoter binding of p65 and of methylation of histone residues permissive of transcription.

  14. Investigation and analysis of single nucleotide polymorphisms in Janus kinase/signal transducer and activator of transcription genes with leukemia.

    PubMed

    Zhong, Yuejiao; Wu, Jianzhong; Chen, Baoan; Ma, Rong; Cao, Haixia; Wang, Zhuo; Cheng, Lu; Ding, Jiahua; Feng, Jifeng

    2012-06-01

    Aberrant activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway may predispose to leukemia due to deregulation of proliferation, differentiation or apoptosis. This study was conducted to investigate whether any association exists between genetic polymorphisms in the JAK2, STAT3 and STAT5 genes and individual susceptibility to leukemia. A case-control study was carried out using a Chinese sample set with 344 cases of leukemia and 346 controls matched by age and ethnicity. Genomic DNA was assayed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) on 13 single nucleotide polymorphisms (SNPs). Genotype analyses showed that two SNPs, namely rs17886724 and rs2293157 located in STAT3 and STAT5, respectively, were significantly associated with leukemia (p < 0.05 for all). Interaction analyses of SNPs (rs17886724|rs2293157; rs11079041| rs2293157) showed that there were inferior associations in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) compared to the control group (0.1 > p > 0.05). Linkage disequilibrium existed between rs11079041 and rs2293157 in both leukemia and control groups (r(2) = 0.7). The haplotypes displayed significant association between rs11079041 and rs2293157 in both leukemia and control groups (p < 0.05). The accuracy rate of the support vector machine (SVM) classification model in making a prediction of leukemia was 97%. The results indicated that STAT3 and STAT5 gene SNPs may be prognostic of leukemia.

  15. Trm1p, a Zn(II)₂Cys₆-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris.

    PubMed

    Sahu, Umakant; Krishna Rao, Kamisetty; Rangarajan, Pundi N

    2014-08-15

    The zinc finger transcription factors Mxr1p and Rop are key regulators of methanol metabolism in the methylotrophic yeast, Pichia pastoris, while Trm1p and Trm2p regulate methanol metabolism in Candida boidinii. Here, we demonstrate that Trm1p is essential for the expression of genes of methanol utilization (mut) pathway in P. pastoris as well. Expression of AOXI and other genes of mut pathway is severely compromised in P. pastoris ΔTrm1 strain resulting in impaired growth on media containing methanol as the sole source of carbon. Trm1p localizes to the nucleus of cells cultured on glucose or methanol. The zinc finger domain of Mxr1p but not Trm1p binds to AOXI promoter sequences in vitro, indicating that these two positive regulators act by different mechanisms. We conclude that both Trm1p and Mxr1p are essential for the expression of genes of mut pathway in P. pastoris and the mechanism of transcriptional regulation of mut pathway may be similar in P. pastoris and C. boidinii.

  16. Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor.

    PubMed

    Kumagai, Takeshi; Aratsu, Yusuke; Sugawara, Ryosuke; Sasaki, Takamitsu; Miyairi, Shinichi; Nagata, Kiyoshi

    2016-04-01

    Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an anti-inflammatory and an anti-viral for the treatment of hepatitis, influenza, and various types of inflammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of the biologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA) inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- and rifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-Lan-Gen, activated CYP3A4 gene transcription through the activation of the human PXR.

  17. Transcriptional regulation of the uncoupling protein-1 gene.

    PubMed

    Villarroya, Francesc; Peyrou, Marion; Giralt, Marta

    2017-03-01

    Regulated transcription of the uncoupling protein-1 (UCP1) gene, and subsequent UCP1 protein synthesis, is a hallmark of the acquisition of the differentiated, thermogenically competent status of brown and beige/brite adipocytes, as well as of the responsiveness of brown and beige/brite adipocytes to adaptive regulation of thermogenic activity. The 5' non-coding region of the UCP1 gene contains regulatory elements that confer tissue specificity, differentiation dependence, and neuro-hormonal regulation to UCP1 gene transcription. Two main regions-a distal enhancer and a proximal promoter region-mediate transcriptional regulation through interactions with a plethora of transcription factors, including nuclear hormone receptors and cAMP-responsive transcription factors. Co-regulators, such as PGC-1α, play a pivotal role in the concerted regulation of UCP1 gene transcription. Multiple interactions of transcription factors and co-regulators at the promoter region of the UCP1 gene result in local chromatin remodeling, leading to activation and increased accessibility of RNA polymerase II and subsequent gene transcription. Moreover, a commonly occurring A-to-G polymorphism in close proximity to the UCP1 gene enhancer influences the extent of UCP1 gene transcription. Notably, it has been reported that specific aspects of obesity and associated metabolic diseases are associated with human population variability at this site. On another front, the unique properties of the UCP1 promoter region have been exploited to develop brown adipose tissue-specific gene delivery tools for experimental purposes.

  18. 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

  19. LATERAL ROOT PRIMORDIA 1 of maize acts as a transcriptional activator in auxin signalling downstream of the Aux/IAA gene rootless with undetectable meristem 1.

    PubMed

    Zhang, Yanxiang; von Behrens, Inga; Zimmermann, Roman; Ludwig, Yvonne; Hey, Stefan; Hochholdinger, Frank

    2015-07-01

    Only little is known about target genes of auxin signalling downstream of the Aux/IAA-ARF module. In the present study, it has been demonstrated that maize lateral root primordia 1 (lrp1) encodes a transcriptional activator that is directly regulated by the Aux/IAA protein ROOTLESS WITH UNDETECTABLE MERISTEM 1 (RUM1). Expression of lrp1 is confined to early root primordia and meristems and is auxin-inducible. Based on its primary protein structure, LRP1 is predicted to be a transcription factor. This notion is supported by exclusive LRP1 localization in the nucleus and its ability to activate downstream gene activity. Based on the observation that lrp1 transcription is completely repressed in the semi-dominant gain of function mutant rum1, it was demonstrated that the lrp1 promoter is a direct target of RUM1 proteins. Subsequently, promoter activation assays indicated that RUM1 represses the expression of a GFP reporter fused to the native promoter of lrp1. Constitutive repression of lrp1 in rum1 mutants is a consequence of the stability of mutated rum1 proteins which cannot be degraded by the proteasome and thus constitutively bind to the lrp1 promoter and repress transcription. Taken together, the repression of the transcriptional activator lrp1 by direct binding of RUM1 to its promoter, together with specific expression of lrp1 in root meristems, suggests a function in maize root development via the RUM1-dependent auxin signalling pathway.

  20. Elucidation of major contributors involved in nitrogen removal and transcription level of nitrogen-cycling genes in activated sludge from WWTPs

    PubMed Central

    Che, You; Liang, Peixin; Gong, Ting; Cao, Xiangyu; Zhao, Ying; Yang, Chao; Song, Cunjiang

    2017-01-01

    We investigated nitrogen-cycle bacterial communities in activated sludge from 8 municipal wastewater treatment plants (WWTPs). Redundancy analyses (RDA) showed that temperature was the most significant driving force in shaping microbial community structure, followed by influent NH4+ and total nitrogen (TN). The diversity of ammonia oxidizing and nitrite reducing bacteria were investigated by the construction of amoA, nirS and nirK gene clone libraries. Phylogenetic analysis indicated that Thauera and Mesorhizobium were the predominant nitrite reducing bacteria, and Nitrosomonas was the only detected ammonia oxidizing bacteria in all samples. Quantification of transcription level of nirS and nirK genes indicated that nirS-type nitrite reducing bacteria played the dominant roles in nitrite reduction process. Transcription level of nirS gene positively correlated with influent NH4+ and TN significantly, whereas inversely linked with hydraulic retention time. Temperature had a strong positive correlation to transcription level of amoA gene. Overall, this study deepened our understanding of the major types of ammonia oxidizing and nitrite reducing bacteria in activated sludge of municipal WWTPs. The relationship between transcription level of nitrogen-cycle genes and operational or environmental variables of WWTPs revealed in this work could provide guidance for optimization of operating parameters and improving the performance of nitrogen removal. PMID:28294196

  1. Elucidation of major contributors involved in nitrogen removal and transcription level of nitrogen-cycling genes in activated sludge from WWTPs

    NASA Astrophysics Data System (ADS)

    Che, You; Liang, Peixin; Gong, Ting; Cao, Xiangyu; Zhao, Ying; Yang, Chao; Song, Cunjiang

    2017-03-01

    We investigated nitrogen-cycle bacterial communities in activated sludge from 8 municipal wastewater treatment plants (WWTPs). Redundancy analyses (RDA) showed that temperature was the most significant driving force in shaping microbial community structure, followed by influent NH4+ and total nitrogen (TN). The diversity of ammonia oxidizing and nitrite reducing bacteria were investigated by the construction of amoA, nirS and nirK gene clone libraries. Phylogenetic analysis indicated that Thauera and Mesorhizobium were the predominant nitrite reducing bacteria, and Nitrosomonas was the only detected ammonia oxidizing bacteria in all samples. Quantification of transcription level of nirS and nirK genes indicated that nirS-type nitrite reducing bacteria played the dominant roles in nitrite reduction process. Transcription level of nirS gene positively correlated with influent NH4+ and TN significantly, whereas inversely linked with hydraulic retention time. Temperature had a strong positive correlation to transcription level of amoA gene. Overall, this study deepened our understanding of the major types of ammonia oxidizing and nitrite reducing bacteria in activated sludge of municipal WWTPs. The relationship between transcription level of nitrogen-cycle genes and operational or environmental variables of WWTPs revealed in this work could provide guidance for optimization of operating parameters and improving the performance of nitrogen removal.

  2. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    PubMed

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV.

  3. Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor

    PubMed Central

    Ares, Miguel A.; Fernández-Vázquez, José L.; Pacheco, Sabino; Martínez-Santos, Verónica I.; Jarillo-Quijada, Ma. Dolores; Torres, Javier; Alcántar-Curiel, María D.; González-y-Merchand, Jorge A.; De la Cruz, Miguel A.

    2017-01-01

    Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae. PMID:28278272

  4. Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

    PubMed

    Ares, Miguel A; Fernández-Vázquez, José L; Pacheco, Sabino; Martínez-Santos, Verónica I; Jarillo-Quijada, Ma Dolores; Torres, Javier; Alcántar-Curiel, María D; González-Y-Merchand, Jorge A; De la Cruz, Miguel A

    2017-01-01

    Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

  5. A Model for Aryl Hydrocarbon Receptor-Activated Gene Expression Shows Potency and Efficacy Changes and Predicts Squelching Due to Competition for Transcription Co-Activators

    PubMed Central

    Simon, Ted W.; Budinsky, Robert A.; Rowlands, J. Craig

    2015-01-01

    A stochastic model of nuclear receptor-mediated transcription was developed based on activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and subsequent binding the activated AHR to xenobiotic response elements (XREs) on DNA. The model was based on effects observed in cells lines commonly used as in vitro experimental systems. Following ligand binding, the AHR moves into the cell nucleus and forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT). In the model, a requirement for binding to DNA is that a generic coregulatory protein is subsequently bound to the AHR-ARNT dimer. Varying the amount of coregulator available within the nucleus altered both the potency and efficacy of TCDD for inducing for transcription of CYP1A1 mRNA, a commonly used marker for activation of the AHR. Lowering the amount of available cofactor slightly increased the EC50 for the transcriptional response without changing the efficacy or maximal response. Further reduction in the amount of cofactor reduced the efficacy and produced non-monotonic dose-response curves (NMDRCs) at higher ligand concentrations. The shapes of these NMDRCs were reminiscent of the phenomenon of squelching. Resource limitations for transcriptional machinery are becoming apparent in eukaryotic cells. Within single cells, nuclear receptor-mediated gene expression appears to be a stochastic process; however, intercellular communication and other aspects of tissue coordination may represent a compensatory process to maintain an organism’s ability to respond on a phenotypic level to various stimuli within an inconstant environment. PMID:26039703

  6. Effect of selenium deficiency on gene transcription

    SciTech Connect

    Christensen, M.J.; Burgener, K.W. )

    1991-03-11

    To investigate the general effects of dietary selenium (Se) deficiency on gene transcription, weanling male Sprague-Dawley rats were fed a basal Se-deficient Torula yeast-based diet or the same diet supplemented with 0.5 ppm Se as sodium selenite for 40 days. At that time three rats in each dietary group were sacrificed. Livers were excised and divided into two portions for isolation of nuclei and for assay of cytosolic Se-glutathione peroxidase (Se-GPX) activity. Se-GPX activity was 279 {plus minus} 4 (mean {plus minus} SEM) mUnits/mg protein in Se-adequate livers, and 10 {plus minus} 2 mUnits/mg protein in Se-deficient livers. One aliquot of nuclei from each dietary group was used in a run-on transcription assay, employing {alpha}-{sup 32}P-UTP to label nascent transcripts. Equal quantities of radioactivity from these nuclei were hybridized with cDNA probes bound to nitrocellulose. Message bound to each probe was quantitated by laser densitometry of autoradiographs, and by scintillation counting of dot blotted nitrocellulose. Transcription of most genes tested, including Se-GPX, was not significantly affected by dietary Se intake. However, the amount of hybridization to a murine oncogene probe (v-fos) was increased in Se deficiency.

  7. dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes.

    PubMed

    Lloret-Llinares, Marta; Pérez-Lluch, Sílvia; Rossell, David; Morán, Tomás; Ponsa-Cobas, Joan; Auer, Herbert; Corominas, Montserrat; Azorín, Fernando

    2012-10-01

    H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol llo(ser5) form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription.

  8. Temporal Dissection of Rate Limiting Transcriptional Events Using Pol II ChIP and RNA Analysis of Adrenergic Stress Gene Activation

    PubMed Central

    Morris, Daniel P.; Lei, Beilei; Longo, Lawrence D.; Bomsztyk, Karol; Schwinn, Debra A.; Michelotti, Gregory A.

    2015-01-01

    In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the generality of genetic control subsequent to RNA polymerase II (Pol II) recruitment. In this report, we use Pol II Chromatin Immunoprecipitation to investigate relationships between the mechanistic events controlling immediate early gene (IEG) activation following stimulation of the α1a-Adrenergic Receptor expressed in rat-1 fibroblasts. We validate our Pol II ChIP assay by comparison to major transcriptional events assessable by microarray and PCR analysis of precursor and mature mRNA. Temporal analysis of Pol II density suggests that reduced proximal pausing often enhances gene expression and was essential for Nr4a3 expression. Nevertheless, for Nr4a3 and several other genes, proximal pausing delayed the time required for initiation of productive elongation, consistent with a role in ensuring transcriptional fidelity. Arrival of Pol II at the 3’ cleavage site usually correlated with increased polyadenylated mRNA; however, for Nfil3 and probably Gprc5a expression was delayed and accompanied by apparent pre-mRNA degradation. Intragenic pausing not associated with polyadenylation was also found to regulate and delay Gprc5a expression. Temporal analysis of Nr4a3, Dusp5 and Nfil3 shows that transcription of native IEG genes can proceed at velocities of 3.5 to 4 kilobases/min immediately after activation. Of note, all of the genes studied here also used increased Pol II recruitment as an important regulator of expression. Nevertheless, the generality of co-transcriptional regulation during IEG activation suggests temporal and integrated analysis will often be necessary to distinguish causative from potential rate limiting mechanisms. PMID:26244980

  9. Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene.

    PubMed

    Chen, Yuting; Lu, Wenqing; Gao, Na; Long, Yi; Shao, Yanjiao; Liu, Meizhen; Chen, Huaqing; Ye, Shixin; Ma, Xueyun; Liu, Mingyao; Li, Dali

    2017-02-01

    The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases (TALENs) technology to generate Leptin receptor (Lepr) knockout rats on the Sprague Dawley (SD) genetic background. Through direct injection of in vitro transcribed mRNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.

  10. Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

    PubMed

    Chan, Chien-Pin; Tsai, Yao-Ting; Chen, Yao-Li; Hsu, Yu-Wen; Tseng, Joseph T; Chuang, Hung-Yi; Shiurba, Robert; Lee, Mei-Hsien; Wang, Jaw-Yuan; Chang, Wei-Chiao

    2015-02-01

    Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 μM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture.

  11. Regulation of photoreceptor gene transcription via a highly conserved transcriptional regulatory element by vsx gene products

    PubMed Central

    Pan, Yi; Comiskey, Daniel F.; Kelly, Lisa E.; Chandler, Dawn S.

    2016-01-01

    Purpose The photoreceptor conserved element-1 (PCE-1) sequence is found in the transcriptional regulatory regions of many genes expressed in photoreceptors. The retinal homeobox (Rx or Rax) gene product functions by binding to PCE-1 sites. However, other transcriptional regulators have also been reported to bind to PCE-1. One of these, vsx2, is expressed in retinal progenitor and bipolar cells. The purpose of this study is to identify Xenopus laevis vsx gene products and characterize vsx gene product expression and function with respect to the PCE-1 site. Methods X. laevis vsx gene products were amplified with PCR. Expression patterns were determined with in situ hybridization using whole or sectioned X. laevis embryos and digoxigenin- or fluorescein-labeled antisense riboprobes. DNA binding characteristics of the vsx gene products were analyzed with electrophoretic mobility shift assays (EMSAs) using in vitro translated proteins and radiolabeled oligonucleotide probes. Gene transactivation assays were performed using luciferase-based reporters and in vitro transcribed effector gene products, injected into X. laevis embryos. Results We identified one vsx1 and two vsx2 gene products. The two vsx2 gene products are generated by alternate mRNA splicing. We verified that these gene products are expressed in the developing retina and that expression resolves into distinct cell types in the mature retina. Finally, we found that vsx gene products can bind the PCE-1 site in vitro and that the two vsx2 isoforms have different gene transactivation activities. Conclusions vsx gene products are expressed in the developing and mature neural retina. vsx gene products can bind the PCE-1 site in vitro and influence the expression of a rhodopsin promoter-luciferase reporter gene. The two isoforms of vsx have different gene transactivation activities in this reporter gene system. PMID:28003732

  12. Cisplatin upregulates Saccharomyces cerevisiae genes involved in iron homeostasis through activation of the iron insufficiency-responsive transcription factor Aft1.

    PubMed

    Kimura, Akiko; Ohashi, Kazuaki; Naganuma, Akira

    2007-02-01

    The response of Saccharomyces cerevisiae to cisplatin was investigated by examining variations in gene expression using cDNA microarrays and confirming the results by reverse transcription polymerase chain reaction (RT-PCR). The mRNA levels of 14 proteins involved in iron homeostasis were shown to be increased by cisplatin. Interestingly, the expression of all 14 genes is known to be regulated by Aft1, a transcription factor activated in response to iron insufficiency. The promoter of one of these genes, FET3, has been relatively well studied, so we performed a reporter assay using the FET3 promoter and showed that an Aft1 binding site in the promoter region is indispensable for induction of transcription by cisplatin. The active domain of Aft1 necessary for activation of the FET3 promoter by cisplatin is identical to the one required for activation by bathophenanthroline sulfonate, an inhibitor of cellular iron uptake. Furthermore, we found that cisplatin inhibits the uptake of (55)Fe(II) into yeast cells. These findings suggest that cisplatin activates Aft1 through the inhibition of iron uptake into the cells, after which the expression of Aft1 target genes involved in iron uptake might be induced.

  13. Exogenous calcium induces tolerance to atrazine stress in Pennisetum seedlings and promotes photosynthetic activity, antioxidant enzymes and psbA gene transcripts.

    PubMed

    Erinle, Kehinde Olajide; Jiang, Zhao; Ma, Bingbing; Li, Jinmei; Chen, Yukun; Ur-Rehman, Khalil; Shahla, Andleeb; Zhang, Ying

    2016-10-01

    Calcium (Ca) has been reported to lessen oxidative damages in plants by upregulating the activities of antioxidant enzymes. However, atrazine mediated reactive oxygen species (ROS) reduction by Ca is limited. This study therefore investigated the effect of exogenously applied Ca on ROS, antioxidants activity and gene transcripts, the D1 protein (psbA gene), and chlorophyll contents in Pennisetum seedlings pre-treated with atrazine. Atrazine toxicity increased ROS production and enzyme activities (ascorbate peroxidase APX, peroxidase POD, Superoxide dismutase SOD, glutathione-S-transferase GST); but decreased antioxidants (APX, POD, and Cu/Zn SOD) and psbA gene transcripts. Atrazine also decreased the chlorophyll contents, but increased chlorophyll (a/b) ratio. Contrarily, Ca application to atrazine pre-treated seedlings lowered the harmful effects of atrazine by reducing ROS levels, but enhancing the accumulation of total chlorophyll contents. Ca-protected seedlings in the presence of atrazine manifested reduced APX and POD activity, whereas SOD and GST activity was further increased with Ca application. Antioxidant gene transcripts that were down-regulated by atrazine toxicity were up-regulated with the application of Ca. Calcium application also resulted in up-regulation of the D1 protein. In conclusion, ability of calcium to reverse atrazine-induced oxidative damage and calcium regulatory role on GST in Pennisetum was presented.

  14. An ortholog of farA of Aspergillus nidulans is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast Yarrowia lipolytica

    SciTech Connect

    Poopanitpan, Napapol; Kobayashi, Satoshi; Fukuda, Ryouichi; Horiuchi, Hiroyuki; Ohta, Akinori

    2010-11-26

    Research highlights: {yields} POR1 is a Yarrowia lipolytica ortholog of farA involved in fatty acid response in A. nidulans. {yields} Deletion of POR1 caused growth defects on fatty acids. {yields} {Delta}por1 strain exhibited defects in the induction of genes involved in fatty acid utilization. -- Abstract: The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y. lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in {beta}-oxidation and peroxisome proliferation by oleate was distinctly diminished in the {Delta}por1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.

  15. A fusion promoter created by a new insertion sequence, IS1490, activates transcription of 2,4,5-trichlorophenoxyacetic acid catabolic genes in Burkholderia cepacia AC1100.

    PubMed Central

    Hübner, A; Hendrickson, W

    1997-01-01

    Transposition and transcriptional activation by insertion sequences in Burkholderia cepacia AC1100 were investigated. Two closely related new elements, IS1413 and IS1490, were identified and characterized. These elements are not highly related to other insertion sequences identified in AC1100 or other B. cepacia isolates. Based on their structures and the sequences of the inverted terminal repeats and the putative transposase protein, the insertion elements (IS elements) are similar to IST2 of Thiobacillus ferrooxidans and several related elements. All the IS elements that have been identified in this strain are found in multiple copies (10 to 40), and they have high-level promoter activity capable of stimulating transcription from a distance up to 500 bp from a target gene. Strain AC1100 was originally isolated after prolonged selection for the ability to utilize the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole carbon source. Three IS elements are located near the first gene of the 2,4,5-T catabolic pathway, tftA. IS1490 inserted 110 bp upstream of tftA and created a fusion promoter responsible for constitutive transcription of the gene. Our results confirm the hypothesis that IS elements play a central role in transcription of 2,4,5-T genes and likely have stimulated rapid evolution of the metabolic pathway. PMID:9098071

  16. Transcriptional regulation by post-transcriptional modification--role of phosphorylation in Sp1 transcriptional activity.

    PubMed

    Chu, Shijian

    2012-10-15

    Sp1 is a ubiquitously expressed transcription factor involved in the regulation of a large number of genes including housekeeping genes as well as actively regulated genes. Although Sp1 was discovered nearly three decades ago, its functional diversity is still not completely understood. One of the ways that make Sp1 versatile in transcriptional regulation is its post-transcriptional modification, which alters Sp1 structure in different cells and at different times. Compared to other types of modifications of the Sp1 protein, phosphorylation has been studied far more extensively. This review focuses on the inducers, pathways, enzymes, and biological effects of Sp1 phosphorylation. Recent data are beginning to reveal the biological significance and universal presence of Sp1 phosphorylation-related cell/molecular responses. Studies in this field provide a quick glance at how a simple chemical modification of a transcription factor could produce significant functional diversity of the protein.

  17. DNA footprint analysis of the transcriptional activator proteins NodD1 and NodD3 on inducible nod gene promoters.

    PubMed Central

    Fisher, R F; Long, S R

    1989-01-01

    The Rhizobium meliloti nodD1 and nodD3 gene products (NodD1 and NodD3) are members of the lysR-nodD gene regulator family. They are functionally distinct in that NodD1 transcriptionally activates other nod genes in the presence of a flavonoid inducer such as luteolin, while NodD3 is capable of activating nod gene expression at high levels in the absence of inducer. NodD1 and NodD3 are DNA-binding proteins which interact with DNA sequences situated upstream of the transcription initiation sites of at least three sets of inducible nod genes. We report the footprinting of NodD1- and NodD3-DNA complexes with both DNase I and the 1,10-phenanthroline-copper ion reagent. NodD1 and NodD3 both interacted with the nodABC, nodFE, and nodH promoters and protected from cleavage an extensive piece of DNA, including the nod box, from approximately -20 to -75 from the transcription start site for each of the three promoters. The constitutively activating protein NodD3 displayed an additional hypersensitive cleavage site in its footprint compared with NodD1. Images PMID:2793828

  18. The transcription factors Pap1 and Prr1 collaborate to activate antioxidant, but not drug tolerance, genes in response to H2O2.

    PubMed

    Calvo, Isabel A; García, Patricia; Ayté, José; Hidalgo, Elena

    2012-06-01

    In response to hydrogen peroxide (H(2)O(2)), the transcription factor Pap1 from Schizosaccharomyces pombe regulates transcription of genes required for adaptation to oxidative stress and for tolerance to toxic drugs. H(2)O(2) induces oxidation of Pap1, its nuclear accumulation and expression of more than fifty Pap1-dependent genes. Oxidation and nuclear accumulation of Pap1 can also be accomplished by genetic inhibition of thioredoxin reductase. Furthermore, genetic alteration of the nuclear export pathway, or mutations in Pap1 nuclear export signal trigger nuclear accumulation of reduced Pap1. We show here that a subset of Pap1-dependent genes, such as those coding for the efflux pump Caf5, the ubiquitin-like protein Obr1 or the dehydrogenase SPCC663.08c, only require nuclear Pap1 for activation, whereas another subset of genes, those coding for the antioxidants catalase, sulfiredoxin or thioredoxin reductase, do need oxidized Pap1 to form a heterodimer with the constitutively nuclear transcription factor Prr1. The ability of Pap1 to bind and activate drug tolerance promoters is independent on Prr1, whereas its affinity for the antioxidant promoters is significantly enhanced upon association with Prr1. This finding suggests that the activation of both antioxidant and drug resistance genes in response to oxidative stress share a common inducer, H(2)O(2), but alternative effectors.

  19. Semax and Pro-Gly-Pro activate the transcription of neurotrophins and their receptor genes after cerebral ischemia.

    PubMed

    Dmitrieva, Veronika G; Povarova, Oksana V; Skvortsova, Veronika I; Limborska, Svetlana A; Myasoedov, Nikolai F; Dergunova, Lyudmila V

    2010-01-01

    Consisting of a fragment of ACTH(4-7) and C-terminal PGP tripeptide, the polypeptide Semax is successfully used for acute stroke therapy. Previous experiments showed rapid induction of Bdnf, Ngf, and TrkB expression in intact rat hippocampus following Semax treatment. To investigate the mRNA expression of neurotrophins and their receptors after treatment with either Semax or PGP, the rat brains were analyzed at three time points following a permanent middle cerebral artery occlusion (pMCAO). We have shown for the first time that both Semax and PGP activate the transcription of neurotrophins and their receptors in the cortex of rats subjected to pMCAO. The profiles of transcription alteration under PGP and Semax treatment were partially overlapped. Semax enhanced the transcription of Bdnf, TrkC, and TrkA 3 h after occlusion, Nt-3 and Ngf 24 h after occlusion, and Ngf 72 h after occlusion. PGP enhanced the transcription of Bdnf and TrkC 3 h after pMCAO and Ngf, TrkB, TrkC, and TrkA 24 h after pMCAO. The analysis of the transcription alterations under PGP and Semax treatment in the cortex of rats without surgery, sham-operated rats and rats subjected to pMCAO revealed that Semax selectively affected the transcription of neurotrophins and their receptors in the ischemic rat cortex, whereas the influence of PGP was mainly unspecific.

  20. Global transcription regulation by DNA topoisomerase I in exponentially growing Saccharomyces cerevisiae cells: activation of telomere-proximal genes by TOP1 deletion.

    PubMed

    Lotito, Luca; Russo, Alessandra; Chillemi, Giovanni; Bueno, Susana; Cavalieri, Duccio; Capranico, Giovanni

    2008-03-21

    To establish the cellular functions of DNA topoisomerase I-B (Top1p) at a global level, we have determined the expression profiles and histone modification patterns affected by TOP1 gene deletion (DeltaTOP1) in Saccharomyces cerevisiae. In exponentially growing cells, DeltaTOP1 specifically increases transcription of telomere-proximal genes and decreases glucose utilization and energy production pathways. Immunoprecipitation data demonstrate that Top1p can bind to and is catalytically active at telomeric DNA repeats, and that both DeltaTOP1 and an inactive Y727F Top1p mutant increase H4 histone acetylation at telomere-proximal regions. Interestingly, while the Y727F mutation has no influence on enzyme recruitment to chromatin sites, it has a marked effect on H4 K16 acetylation at subtelomeric regions. The Top1p mutation also increases H3 histone K4 dimethylation, which has been associated with gene transcription, at 3' termini of subtelomeric genes. No major effect of DeltaTOP1 or mutation was detected on Sir3p recruitment; however, DeltaTOP1 has an effect on transcript levels of genes known to regulate telomeric silencing. Thus, the findings indicate that Top1p activity can favor both a repressed chromatin organization and a reduced gene expression level at telomere-proximal regions in yeast. As telomere-proximal regions are known to be enriched for stress-activated genes, our findings show that Top1p can optimize transcript levels for cell growth in exponentially growing cells under a synthetic medium with glucose.

  1. Transcription dynamics of inducible genes modulated by negative regulations.

    PubMed

    Li, Yanyan; Tang, Moxun; Yu, Jianshe

    2015-06-01

    Gene transcription is a stochastic process in single cells, in which genes transit randomly between active and inactive states. Transcription of many inducible genes is also tightly regulated: It is often stimulated by extracellular signals, activated through signal transduction pathways and later repressed by negative regulations. In this work, we study the nonlinear dynamics of the mean transcription level of inducible genes modulated by the interplay of the intrinsic transcriptional randomness and the repression by negative regulations. In our model, we integrate negative regulations into gene activation process, and make the conventional assumption on the production and degradation of transcripts. We show that, whether or not the basal transcription is temporarily terminated when cells are stimulated, the mean transcription level grows in the typical up and down pattern commonly observed in immune response genes. With the help of numerical simulations, we clarify the delicate impact of the system parameters on the transcription dynamics, and demonstrate how our model generates the distinct temporal gene-induction patterns in mouse fibroblasts discerned in recent experiments.

  2. RNA Activation of the Vascular Endothelial Growth Factor Gene (VEGF) Promoter by Double-Stranded RNA and Hypoxia: Role of Noncoding VEGF Promoter Transcripts

    PubMed Central

    Wagner, Kay-Dietrich; Hofman, Paul; Van Obberghen, Emmanuel

    2016-01-01

    RNA activation (RNAa) is a gene regulation process in which promoter-targeted short double-stranded RNAs (dsRNAs) or microRNAs (miRs) induce target gene expression at the transcriptional level. Here, we investigate the presence of cryptic promoter transcripts within the VEGF promoter. Single-strand sense and antisense noncoding vascular endothelial growth factor (NcVEGF) promoter transcripts are identified, and their respective expression is studied in cells transfected with a VEGF promoter targeted dsRNA, namely, dsVEGF706, in hypoxic cells and in human malignant lung tissues. Interestingly, in dsVEGF706-transfected, as well as in hypoxic cells, NcVEGF expression levels increase coordinately with coding VEGF expression. Ago2 interaction with both sense and antisense NcVEGFs is increased in hypoxic cells, whereas in dsVEGF706-transfected cells, Ago2 and the antisense strand of the dsRNA interact specifically with the sense NcVEGF transcript. Furthermore, both dsVEGF706 and ectopic NcVEGF transcripts are able to activate the VEGF promoter endogenously present or in a reporter construct. Finally, using small interfering RNA targeting Ago2, we show that RNAa plays a role in the maintenance of increased VEGF and NcVEGF expression after hypoxia. Given the central role of VEGF in major human diseases, including cancer, this novel molecular mechanism is poised to reveal promising possibilities for therapeutic interventions. PMID:26976645

  3. The "fourth dimension" of gene transcription.

    PubMed

    O'Malley, Bert W

    2009-05-01

    The three dimensions of space provide our relationship to position on the earth, but the fourth dimension of time has an equally profound influence on our lives. Everything from light and sound to weather and biology operate on the principle of measurable temporal periodicity. Consequently, a wide variety of time clocks affect all aspects of our existence. The annual (and biannual) cycles of activity, metabolism, and mating, the monthly physiological clocks of women and men, and the 24-h diurnal rhythms of humans are prime examples. Should it be surprising to us that the fourth dimension also impinges upon gene expression and that the genome itself is regulated by the fastest running of all biological clocks? Recent evidence substantiates the existence of such a ubiquitin-dependent transcriptional clock that is based upon the activation and destruction of transcriptional coactivators.

  4. An invariant aspartic acid in the DNA glycosylase domain of DEMETER is necessary for transcriptional activation of the imprinted MEDEA gene

    PubMed Central

    Choi, Yeonhee; Harada, John J.; Goldberg, Robert B.; Fischer, Robert L.

    2004-01-01

    Helix-hairpin-helix DNA glycosylases are typically small proteins that initiate repair of DNA by excising damaged or mispaired bases. An invariant aspartic acid in the active site is involved in catalyzing the excision reaction. Replacement of this critical residue with an asparagine severely reduces catalytic activity but preserves enzyme stability and structure. The Arabidopsis DEMETER (DME) gene encodes a large 1,729-aa polypeptide with a 200-aa DNA glycosylase domain. DME is expressed primarily in the central cell of the female gametophyte. DME activates maternal allele expression of the imprinted MEDEA (MEA) gene in the central cell and is required for seed viability. We mutated the invariant aspartic acid at position 1304 in DME to asparagine (D1304N) to determine whether the catalytic activity of the DNA glycosylase domain is required for DME function in vivo. Transgenes expressing wild-type DME in the central cell rescue seed abortion caused by a mutation in the endogenous DME gene and activate maternal MEA:GFP transcription. However, transgenes expressing the D1304N mutant DME do not rescue seed abortion or activate maternal MEA:GFP transcription. Whereas ectopic expression of the wild-type DME polypeptide in pollen is sufficient to activate ectopic paternal MEA and MEA:GUS expression, equivalent expression of the D1304N mutant DME in pollen failed to do so. These results show that the conserved aspartic acid residue is necessary for DME to function in vivo and suggest that an active DNA glycosylase domain, normally associated with DNA repair, promotes gene transcription that is essential for gene imprinting. PMID:15128940

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

    PubMed

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

    2014-08-01

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

  6. Retroviral insertional activation in a herpesvirus: transcriptional activation of US genes by an integrated long terminal repeat in a Marek's disease virus clone.

    PubMed Central

    Jones, D; Brunovskis, P; Witter, R; Kung, H J

    1996-01-01

    Insertional activation of host proto-oncogenes has been recognized as a basic mechanism by which nonacute retroviruses induce cancer. Our previous work has demonstrated that retroviruses can efficiently integrate into DNA virus genomes. Specifically, coinfection of cultured fibroblasts with a chicken herpesvirus, Marek's disease virus (MDV), and a chicken retrovirus results in frequent stable retroviral insertions into the herpesvirus genome. Such insertions could alter the expression of herpesvirus genes, possibly resulting in novel phenotypic properties. In this article, we report the characterization of a replication-competent clone of MDV with integrated retroviral sequences. This virus was isolated from a chicken following injection of fibroblasts coinfected with MDV and the retrovirus, reticuloendotheliosis virus. Transcripts originating from the reticuloendotheliosis virus long terminal repeat promoters were found to encode the adjoining MDV genes, SORF2, US1, and US10. This virus replicates well in culture but has an unusual phenotype in chickens, characterized by an attenuated virulence which produces no nerve lesions but, rather, severe thymic atrophy. While the causal relationship between the insertion and the observed phenotypes remains to be established, our data provide the first evidence of retroviral insertional activation of herpesvirus genes. PMID:8642673

  7. 1,25-dihydroxyvitamin D3 suppresses renin gene transcription by blocking the activity of the cyclic AMP response element in the renin gene promoter.

    PubMed

    Yuan, Weihua; Pan, Wei; Kong, Juan; Zheng, Wei; Szeto, Frances L; Wong, Kari E; Cohen, Ronald; Klopot, Anna; Zhang, Zhongyi; Li, Yan Chun

    2007-10-12

    We have shown that 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) down-regulates renin expression. To explore the molecular mechanism, we analyzed the mouse Ren-1c gene promoter by luciferase reporter assays. Deletion analysis revealed two DNA fragments from -2,725 to -2,647 (distal fragment) and from -117 to +6 (proximal fragment) that are sufficient to mediate the repression. Mutation of the cAMP response element (CRE) in the distal fragment blunted forskolin stimulation as well as 1,25(OH)(2)D(3) inhibition of the transcriptional activity, suggesting the involvement of CRE in 1,25(OH)(2)D(3)-induced suppression. EMSA revealed that 1,25(OH)(2)D(3) markedly inhibited nuclear protein binding to the CRE in the promoter. ChIP and GST pull-down assays demonstrated that liganded VDR blocked the binding of CREB to the CRE by directly interacting with CREB with the ligand-binding domain, and the VDR-mediated repression can be rescued by CREB, CBP, or p300 overexpression. These data indicate that 1,25(OH)(2)D(3) suppresses renin gene expression at least in part by blocking the formation of CRE-CREB-CBP complex.

  8. Role of Calcineurin, hnRNPA2 and Akt in Mitochondrial Respiratory Stress-Mediated Transcription Activation of Nuclear Gene Targets

    PubMed Central

    Guha, Manti; Tang, Weigang; Sondheimer, Neal; Avadhani, Narayan G.

    2010-01-01

    Pathophysiological conditions causing mitochondrial dysfunction and altered transmembrane potential (Δψm) initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a variety of mammalian cells. An increase in the cytosolic Ca2+ [Ca2+]c as part of this signaling cascade activates Ca2+ responsive phosphatase, Calcineurin (Cn). Activation of IGF1R accompanied by increased glycolysis, invasiveness, and resistance to apoptosis are phenotypic hallmarks of C2C12 rhabdomyoblast cells subjected to this stress. The signaling is associated with activation and increased nuclear translocation of a number of transcription factors including a novel NFκB (cRel: p50) pathway, NFAT, CREB and C/EBPδ. This culminates in the upregulation of a number of nuclear genes including Cathepsin L, RyR1, Glut4 and Akt1. We observed that stress regulated transcription activation of nuclear genes involves a cooperative interplay between NFκB (cRel:p50), C/EBPδ, CREB, NFAT. Our results show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein, hnRNPA2 as a transcriptional co-activator. We report here that mitochondrial stress leads to induced expression and activation of serine threonine kinase Akt1. Interestingly, we observe that Akt1 phosphorylates hnRNPA2 under mitochondrial stress conditions, which is a crucial step for the recruitment of this coactivator to the stress target promoters and culmination in mitochondrial stress-mediated transcription activation of target genes. We propose that mitochondrial stress plays an important role in tumor progression and emergence of invasive phenotypes. PMID:20153290

  9. The Rel/NF-κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity

    PubMed Central

    Chang, Tammy T.; Walther, Isabelle; Li, Chai-Fei; Boonyaratanakornkit, Jim; Galleri, Grazia; Meloni, Maria Antonia; Pippia, Proto; Cogoli, Augusto; Hughes-Fulford, Millie

    2012-01-01

    This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in μg. Immunosuppression during spaceflight is a major barrier to safe, long-term human space habitation and travel. The goals of these experiments were to prove that μg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti-CD28 on board the ISS. An on-board centrifuge was used to generate a 1g simultaneous control to isolate the effects of μg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that μg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down-regulated in μg. Importantly, several key immediate early genes were inhibited in μg. In particular, transactivation of Rel/NF-κB, CREB, and SRF gene targets were down-regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in μg and upon anti-CD3/anti-CD28 stimulation in simulated μg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in μg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that μg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes. PMID:22750545

  10. Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection

    PubMed Central

    Flavell, Steven W.; Kim, Tae-Kyung; Gray, Jesse M.; Harmin, David A.; Hemberg, Martin; Hong, Elizabeth J.; Markenscoff-Papadimitriou, Eirene; Bear, Daniel M.; Greenberg, Michael E.

    2009-01-01

    SUMMARY Although many transcription factors are known to control important aspects of neural development, the genome-wide programs that are directly regulated by these factors are not known. We have characterized the genetic program that is activated by MEF2, a key regulator of activity-dependent synapse development. These MEF2 target genes have diverse functions at synapses, revealing a broad role for MEF2 in synapse development. Several of the MEF2 targets are mutated in human neurological disorders including epilepsy and autism-spectrum disorders, suggesting that these disorders may be caused by disruption of an activity-dependent gene program that controls synapse development. Our analyses also reveal that neuronal activity promotes alternative polyadenylation site usage at many of the MEF2 target genes, leading to the production of truncated mRNAs that may have different functions than their full-length counterparts. Taken together, these analyses suggest that the ubiquitously expressed transcription factor MEF2 regulates an intricate transcriptional program in neurons that controls synapse development. PMID:19109909

  11. Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase-neo cassette located between the murine beta-major and beta-minor globin genes.

    PubMed

    Kaufman, R M; Lu, Z H; Behl, R; Holt, J M; Ackers, G K; Ley, T J

    2001-07-01

    For the treatment of beta-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integration-based gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the beta6 position of the mouse beta-major globin gene (beta(6I)) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies. The marked beta-major gene can be tracked at the level of DNA, RNA, and protein, allowing investigation of the impact of a retained phosphoglycerate kinase (PGK)-neo cassette located between the mutant beta-major and beta-minor globin genes on expression of these 2 neighboring genes. Although the PGK-neo cassette was expressed at high levels in adult erythroid cells, the abundance of the beta(6I) mRNA was indistinguishable from that of the wild-type counterpart in bone marrow cells. Similarly, the output from the beta-minor globin gene was also normal. Therefore, in this specific location, the retained, transcriptionally active PGK-neo cassette does not disrupt the regulated expression of the adult beta-globin genes. (Blood. 2001;98:65-73)

  12. Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum

    PubMed Central

    Liu, Zhiqin; Shi, Lanping; Liu, Yanyan; Tang, Qian; Shen, Lei; Yang, Sheng; Cai, Jinsen; Yu, Huanxin; Wang, Rongzhang; Wen, Jiayu; Lin, Youquan; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; He, Shuilin

    2015-01-01

    The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper. PMID:26442088

  13. Complementary Activities of TELOMERE REPEAT BINDING Proteins and Polycomb Group Complexes in Transcriptional Regulation of Target Genes[OPEN

    PubMed Central

    Hartwig, Benjamin; James, Geo Velikkakam

    2016-01-01

    In multicellular organisms, Polycomb Repressive Complex 1 (PRC1) and PRC2 repress target genes through histone modification and chromatin compaction. Arabidopsis thaliana mutants strongly compromised in the pathway cannot develop differentiated organs. LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is so far the only known plant PRC1 component that directly binds to H3K27me3, the histone modification set by PRC2, and also associates genome-wide with trimethylation of lysine 27 of histone H3 (H3K27me3). Surprisingly, lhp1 mutants show relatively mild phenotypic alterations. To explain this paradox, we screened for genetic enhancers of lhp1 mutants to identify novel components repressing target genes together with, or in parallel to, LHP1. Two enhancing mutations were mapped to TELOMERE REPEAT BINDING PROTEIN1 (TRB1) and its paralog TRB3. We show that TRB1 binds to thousands of genomic sites containing telobox or related cis-elements with a significant increase of sites and strength of binding in the lhp1 background. Furthermore, in combination with lhp1, but not alone, trb1 mutants show increased transcription of LHP1 targets, such as floral meristem identity genes, which are more likely to be bound by TRB1 in the lhp1 background. By contrast, expression of a subset of LHP1-independent TRB1 target genes, many involved in primary metabolism, is decreased in the absence of TRB1 alone. Thus, TRB1 is a bivalent transcriptional modulator that maintains downregulation of Polycomb Group (PcG) target genes in lhp1 mutants, while it sustains high expression of targets that are regulated independently of PcG. PMID:26721861

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

    PubMed Central

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

    2012-01-01

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

  15. RmpA2, an activator of capsule biosynthesis in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the transcriptional level.

    PubMed

    Lai, Yi-Chyi; Peng, Hwei-Ling; Chang, Hwan-You

    2003-02-01

    The rmpA2 gene, which encodes an activator for capsular polysaccharide (CPS) synthesis, was isolated from a 200-kb virulence plasmid of Klebsiella pneumoniae CG43. Based on the sequence homology with LuxR at the carboxyl-terminal DNA-binding motif, we hypothesized that RmpA2 exerts its effect by activating the expression of cps genes that are responsible for CPS biosynthesis. Two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes, were constructed and were found to be activated in the presence of multicopy rmpA2. The activation is likely due to direct binding of RmpA2 to the cps gene promoter through its C-terminal DNA binding motif. Moreover, the loss of colony mucoidy in a K. pneumoniae strain deficient in RcsB, a regulator for cps gene expression, could be recovered by complementing the strain with a multicopy plasmid carrying rmpA2. The CPS production in Lon protease-deficient K. pneumoniae significantly increased, and the effect was accompanied by an increase of RmpA2 stability. The expression of the rmpA2 gene was negatively autoregulated and could be activated when the organism was grown in M9 minimal medium. An IS3 element located upstream of the rmpA2 was required for the full activation of the rmpA2 promoter. In summary, our results suggest that the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 is autoregulated and under the control of Lon protease.

  16. Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo

    PubMed Central

    Rijal, Keshab; Maraia, Richard J.

    2016-01-01

    The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease. PMID:27518095

  17. Disruption of CLOCK-BMAL1 Transcriptional Activity Is Responsible for Aryl Hydrocarbon Receptor–Mediated Regulation of Period1 Gene

    PubMed Central

    Xu, Can-Xin; Krager, Stacey L.; Liao, Duan-Fang; Tischkau, Shelley A.

    2010-01-01

    The aryl hydrocarbon receptor (AhR) is a period-aryl hydrocarbon receptor nuclear transporter-simple minded domain transcription factor that shares structural similarity with circadian clock genes and readily interacts with components of the molecular clock. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters behavioral circadian rhythms and represses the Period1 (Per1) gene in murine hematopoietic stem and progenitor cells. Per1 expression is driven by circadian locomotor activity cycles kaput-brain muscle ARNT-like (CLOCK-BMAL1)–dependent activation of Eboxes in the Per1 promoter. We hypothesized that the effects of AhR activation on the circadian clock are mediated by disruption of CLOCK-BMAL1 function and subsequent Per1 gene suppression. Effects of AhR activation on rhythmic Per1 transcripts were examined in livers of mice after treatment with the AhR agonist, TCDD; the molecular mechanisms of Per1 repression by AhR were determined in hepatoma cells using TCDD and β-napthoflavone as AhR activators. This study reports, for the first time, that AhR activation by TCDD alters the Per1 rhythm in the mouse liver and that Per1 gene suppression depends upon the presence of AhR. Furthermore, AhR interaction with BMAL1 attenuates CLOCK-BMAL1 activity and decreases CLOCK binding at Ebox1 and Ebox3 in the Per1 promoter. Taken together, these data suggest that AhR activation represses Per1 through disrupting CLOCK-BMAL1 activity, producing dysregulation of rhythmic Per1 gene expression. These data define alteration of the Per1 rhythm as novel signaling events downstream of AhR activation. Downregulation of Per1 could contribute to metabolic disease, cancer, and other detrimental effects resulting from exposure to certain environmental pollutants. PMID:20106950

  18. hrpL activates Erwinia amylovora hrp gene transcription and is a member of the ECF subfamily of sigma factors.

    PubMed Central

    Wei, Z M; Beer, S V

    1995-01-01

    hrpL of Erwinia amylovora Ea321 encodes a 21.7-kDa regulatory protein, similar to members of the ECF (extra cytoplasmic functions) subfamily of eubacterial RNA polymerase sigma factors. hrpL is a single-gene operon in complementation group VI of the E. amylovora hrp gene cluster. Its product is required by Ea321 to elicit the hypersensitive response (HR) and to cause disease. HrpL controls the expression of five independent hrp loci, including hrpN, which encodes harpin, a proteinaceous elicitor of the HR. hrpL is environmentally regulated, and its expression is affected by hrpS, another regulatory gene of the hrp gene cluster of E. amylovora. pCPP1078, a multicopy plasmid carrying hrpL, is able to restore HR-eliciting ability to hrpS mutants. A conserved motif was identified upstream of the hrpI and hrpN operons, which are transcriptionally regulated by hrpL. This conserved motif shares a high degree of similarity with other biochemically defined or putative ECF-dependent promoter sequences, including sequences upstream of Streptomyces coelicolor dagA P2, Pseudomonas aeruginosa algD, Pseudomonas syringae pv. syringae 61 hrpZ, and P. syringae pv. tomato avrD. In spite of the similarity between the hrpL genes of E. amylovora and P. syringae 61, no functional cross-complementation was observed. PMID:7592386

  19. Methyl-CpG-Binding Protein MeCP2 Represses Sp1-Activated Transcription of the Human Leukosialin Gene When the Promoter Is Methylated

    PubMed Central

    Kudo, Shinichi

    1998-01-01

    Human leukosialin (CD43) is expressed in a cell lineage-specific as well as a differentiation stage-specific fashion. The leukosialin promoter, made up of an Sp1 binding site and a sequence similar to that of an initiator, possesses high transcriptional potential. Previous data have demonstrated that the leukosialin gene is down-regulated in nonproducing cells by DNA methylation. In this paper the repressive mechanism of DNA methylation in expression systems is reported. In vitro DNA methylation with SssI (CpG) methylase of leukosialin-chloramphenicol acetyltransferase (CAT) constructs drastically reduced transcriptional activities in stable transfection systems with the human HeLa and Jurkat cell lines. On the other hand, the transcriptional repression by in vitro methylation was less pronounced in Drosophila melanogaster cells, which lack genomic methylation. In these cells, Sp1 could transactivate equally well both the unmethylated and methylated leukosialin promoter. In order to test whether one of the methyl-CpG-binding proteins, MeCP2, is responsible for transcriptional repression of the leukosialin gene, I isolated the human MeCP2 cDNA (encoding 486 amino acid residues) and expressed it in Drosophila cells. I found that MeCP2 substantially inhibited Sp1-activated transcription when the leukosialin promoter was methylated. The level of repression was directly proportional to the amount of MeCP2 expression vector transfected. Analysis of C-terminal deletion mutants of MeCP2 showed that repressive activity of Sp1 transactivation is localized to the N-terminal region consisting of amino acid residues 1 to 193, which encompass the methyl-binding domain. These results suggest that interference with Sp1 transactivation by MeCP2 is an important factor in the down-regulation of leukosialin gene expression by DNA methylation. PMID:9710633

  20. Transcription activator-like effector nucleases efficiently disrupt the target gene in Iberian ribbed newts (Pleurodeles waltl), an experimental model animal for regeneration.

    PubMed

    Hayashi, Toshinori; Sakamoto, Kousuke; Sakuma, Tetsushi; Yokotani, Naoki; Inoue, Takeshi; Kawaguchi, Eri; Agata, Kiyokazu; Yamamoto, Takashi; Takeuchi, Takashi

    2014-01-01

    Regeneration of a lost tissue in an animal is an important issue. Although regenerative studies have a history of research spanning more than a century, the gene functions underlying regulation of the regeneration are mostly unclear. Analysis of knockout animals is a very powerful tool with which to elucidate gene function. Recently, transcription activator-like effector nucleases (TALENs) have been developed as an effective technique for genome editing. This technique enables gene targeting in amphibians such as newts that were previously impossible. Here we show that newts microinjected with TALEN mRNAs designed for targeting the tyrosinase gene in single-cell stage embryos revealed an albino phenotype. Sequence analysis revealed that the tyrosinase genes were effectively disrupted in these albino newts. Moreover, precise genome alteration was achieved using TALENs and single strand oligodeoxyribonucleotides. Our results suggest that TALENs are powerful tools for genome editing for regenerative research in newts.

  1. Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

    PubMed

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2015-06-15

    High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases.

  2. Thyroid active agents T3 and PTU differentially affect immune gene transcripts in the head kidney of rainbow trout (Oncorynchus mykiss).

    PubMed

    Quesada-García, Alba; Encinas, Paloma; Valdehita, Ana; Baumann, Lisa; Segner, Helmut; Coll, Julio M; Navas, José M

    2016-05-01

    In mammals, numerous reports describe an immunomodulating effect of thyroid-active compounds. In contrast, only few reports have been published on this subject in fish. We previously demonstrated that immune cells of rainbow trout (Oncorhynchus mykiss) possess thyroid hormone receptors (THRs) and that exposure of trout to the thyroid hormone 3,3',5-triiodo-l-thyronine (T3) or the antithyroid drug propylthiouracil (PTU) alters immune cell transcript levels of THR and several immune genes. The present study aims to further characterize the immunomodulating action of thyroid-active compounds in trout immune cells. We report here the use of a custom-designed 60-mer oligo immune-targeted microarray for rainbow trout to analyze the gene expression profiles induced in the head kidney by T3 and PTU. Morphometric analyses of the thyroid showed that PTU exposure increased the size of the epithelial cells, whereas T3 induced no significant effects. Both T3 and PTU had diverse and partly contrasting effects on immune transcript profiles. The strongest differential effects of T3 and PTU on gene expressions were those targeting the Mitogen Associated Protein Kinase (MAPK), NFkB, Natural Killer (NK) and Toll-Like Receptor (TLR) pathways, a number of multipath genes (MPG) such as those encoding pleiotropic transcription factors (atf1, junb, myc), as well as important pro-inflammatory genes (tnfa, tnf6, il1b) and interferon-related genes (ifng, irf10). With these results we show for the first time in a fish species that the in vivo thyroidal status modulates a diversity of immune genes and pathways. This knowledge provides the basis to investigate both mechanisms and consequences of thyroid hormone- and thyroid disruptor-mediated immunomodulation for the immunocompetence of fish.

  3. Duplicate Maize Wrinkled1 Transcription Factors Activate Target Genes Involved in Seed Oil Biosynthesis1[C][W

    PubMed Central

    Pouvreau, Benjamin; Baud, Sébastien; Vernoud, Vanessa; Morin, Valérie; Py, Cyrille; Gendrot, Ghislaine; Pichon, Jean-Philippe; Rouster, Jacques; Paul, Wyatt; Rogowsky, Peter M.

    2011-01-01

    WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs. PMID:21474435

  4. Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

    PubMed

    DeSmet, Marsha L; Fleet, James C

    2017-01-16

    High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH)2D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH)2D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH)2D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH)2D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention.

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

    PubMed

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

    1997-12-01

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

  6. Transcriptionally active immediate-early protein of pseudorabies virus binds to specific sites on class II gene promoters.

    PubMed Central

    Cromlish, W A; Abmayr, S M; Workman, J L; Horikoshi, M; Roeder, R G

    1989-01-01

    In the presence of partially purified pseudorabies virus immediate-early protein, multiple sites of DNase I protection were observed on the adenovirus major late and human hsp 70 promoters. Southwestern (DNA-protein blot) analysis demonstrated that the immediate-early protein bound directly to the sequences contained in these sites. These sequences share only limited homology, differ in their affinities for the immediate-early protein, and are located at different positions on these two promoters. In addition, the site-specific binding of a temperature-sensitive immediate-early protein was eliminated by the same heat treatment which eliminates its transcriptional activating function, whereas the binding of the wild-type protein was unaffected by heat treatment. Thus, site-specific binding requires a functionally active immediate-early protein. Furthermore, immediate-early-protein-dependent in vitro transcription from the major late promoter was preferentially inhibited by oligonucleotides which are homologous to the high-affinity binding sites on the major late or hsp 70 promoters. These observations suggest that transcriptional stimulation by the immediate-early protein involves binding to cis-acting elements. Images PMID:2539489

  7. Characterization of TetD as a transcriptional activator of a subset of genes of the Escherichia coli SoxS/MarA/Rob regulon.

    PubMed

    Griffith, Kevin L; Becker, Stephen M; Wolf, Richard E

    2005-05-01

    In Escherichia coli, SoxS, MarA and Rob form a closely related subset of the AraC/XylS family of positive regulators, sharing approximately 42% amino acid sequence identity over the length of SoxS and the ability to activate transcription of a common set of target genes that provide resistance to redox-cycling compounds and antibiotics. On the basis of its approximately 43% amino acid sequence identity with SoxS, MarA and Rob, TetD, encoded by transposon Tn10, appears to be a fourth member of the subset. However, although its expression has been shown to be negatively regulated by TetC and not inducible by tetracycline, the physiological function of TetD is unknown. Accordingly, in the work presented here, we initiate a molecular characterization of TetD. We show that expression of TetD activates transcription of a subset of the SoxS/MarA/Rob regulon genes and confers resistance to redox-cycling compounds and antibiotics. We show that mutations in the putative TetD binding site of a TetD-activatable promoter and a mutation in the protein's N-terminal DNA recognition helix interfere with transcription activation, thereby indicating that TetD directly activates target gene transcription. Finally, we show that TetD, like SoxS and MarA, is intrinsically unstable; however, unlike SoxS and MarA, TetD is not degraded by Lon or any of the cell's known cytoplasmic ATP-dependent proteases. Thus, we conclude that TetD is a bona fide member of the SoxS/MarA/Rob subfamily of positive regulators.

  8. Chromatin insulation by a transcriptional activator

    PubMed Central

    Sutter, Nathan B.; Scalzo, David; Fiering, Steven; Groudine, Mark; Martin, David I. K.

    2003-01-01

    In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression. PMID:12547916

  9. The SNF5 protein of Saccharomyces cerevisiae is a glutamine- and proline-rich transcriptional activator that affects expression of a broad spectrum of genes.

    PubMed Central

    Laurent, B C; Treitel, M A; Carlson, M

    1990-01-01

    The Saccharomyces cerevisiae SNF5 gene affects expression of both glucose- and phosphate-regulated genes and appears to function in transcription. We report the nucleotide sequence, which predicts that SNF5 encodes a 102,536-dalton protein. The N-terminal third of the protein is extremely rich in glutamine and proline. Mutants carrying a deletion of the coding sequence were viable but grew slowly, indicating that the SNF5 gene is important but not essential. Evidence that SNF5 affects expression of the cell type-specific genes MF alpha 1 and BAR1 at the RNA level extends the known range of SNF5 function. SNF5 is apparently required for expression of a wide variety of differently regulated genes. A bifunctional SNF5-beta-galactosidase fusion protein was localized in the nucleus by immunofluorescence. No DNA-binding activity was detected for SNF5. A LexA-SNF5 fusion protein, when bound to a lexA operator, functioned as a transcriptional activator. Images PMID:2233708

  10. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

    PubMed

    Polstein, Lauren R; Perez-Pinera, Pablo; Kocak, D Dewran; Vockley, Christopher M; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

    2015-08-01

    Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function.

  11. A Complete Set of Nascent Transcription Rates for Yeast Genes

    PubMed Central

    Pelechano, Vicent; Chávez, Sebastián; Pérez-Ortín, José E.

    2010-01-01

    The amount of mRNA in a cell is the result of two opposite reactions: transcription and mRNA degradation. These reactions are governed by kinetics laws, and the most regulated step for many genes is the transcription rate. The transcription rate, which is assumed to be exercised mainly at the RNA polymerase recruitment level, can be calculated using the RNA polymerase densities determined either by run-on or immunoprecipitation using specific antibodies. The yeast Saccharomyces cerevisiae is the ideal model organism to generate a complete set of nascent transcription rates that will prove useful for many gene regulation studies. By combining genomic data from both the GRO (Genomic Run-on) and the RNA pol ChIP-on-chip methods we generated a new, more accurate nascent transcription rate dataset. By comparing this dataset with the indirect ones obtained from the mRNA stabilities and mRNA amount datasets, we are able to obtain biological information about posttranscriptional regulation processes and a genomic snapshot of the location of the active transcriptional machinery. We have obtained nascent transcription rates for 4,670 yeast genes. The median RNA polymerase II density in the genes is 0.078 molecules/kb, which corresponds to an average of 0.096 molecules/gene. Most genes have transcription rates of between 2 and 30 mRNAs/hour and less than 1% of yeast genes have >1 RNA polymerase molecule/gene. Histone and ribosomal protein genes are the highest transcribed groups of genes and other than these exceptions the transcription of genes is an infrequent phenomenon in a yeast cell. PMID:21103382

  12. CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

    NASA Technical Reports Server (NTRS)

    Umayahara, Y.; Ji, C.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast

  13. Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells

    PubMed Central

    Vajravelu, Bathri N.; Moktar, Afsoon; Cao, Pengxiao; Moore, Joseph B.; Bolli, Roberto

    2017-01-01

    Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy. PMID:28355297

  14. Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells.

    PubMed

    Al-Maqtari, Tareq; Hong, Kyung U; Vajravelu, Bathri N; Moktar, Afsoon; Cao, Pengxiao; Moore, Joseph B; Bolli, Roberto

    2017-01-01

    Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy.

  15. Transcription of the protein kinase C-δ gene is activated by JNK through c-Jun and ATF2 in response to the anticancer agent doxorubicin

    PubMed Central

    Min, Byong Wook; Kim, Chang Gun; Ko, Jesang; Lim, Yoongho

    2008-01-01

    Expression of protein kinase C-δ (PKCδ) is up-regulated by apoptosis-inducing stimuli. However, very little is known about the signaling pathways that control PKCδ gene transcription. In the present study, we demonstrate that JNK stimulates PKCδ gene expression via c-Jun and ATF2 in response to the anticancer agent doxorubicin (DXR) in mouse lymphocytic leukemia L1210 cells. Luciferase reporter assays showed that DXR-induced activation of the PKCδ promoter was enhanced by ectopic expression of JNK1, c-Jun, or ATF2, whereas it was strongly reduced by expression of dominant negative JNK1 or by treatment with the JNK inhibitor SP600125. Furthermore, point mutations in the core sequence of the c-Jun/ATF2 binding site suppressed DXR-induced activation of the PKCδ promoter. Our results suggest an additional role for a JNK signaling cascade in DXR-induced PKCδ gene expression. PMID:19116455

  16. Specific domains of FoxD4/5 activate and repress neural transcription factor genes to control the progression of immature neural ectoderm to differentiating neural plate.

    PubMed

    Neilson, Karen M; Klein, Steven L; Mhaske, Pallavi; Mood, Kathy; Daar, Ira O; Moody, Sally A

    2012-05-15

    FoxD4/5, a forkhead transcription factor, plays a critical role in establishing and maintaining the embryonic neural ectoderm. It both up-regulates genes that maintain a proliferative, immature neural ectoderm and down-regulates genes that promote the transition to a differentiating neural plate. We constructed deletion and mutant versions of FoxD4/5 to determine which domains are functionally responsible for these opposite activities, which regulate the critical developmental transition of neural precursors to neural progenitors to differentiating neural plate cells. Our results show that up-regulation of genes that maintain immature neural precursors (gem, zic2) requires the Acidic blob (AB) region in the N-terminal portion of the protein, indicating that the AB is the transactivating domain. Additionally, down-regulation of those genes that promote the transition to neural progenitors (sox) and those that lead to neural differentiation (zic, irx) involves: 1) an interaction with the Groucho co-repressor at the Eh-1 motif in the C-terminus; and 2) sequence downstream of this motif. Finally, the ability of FoxD4/5 to induce the ectopic expression of neural precursor genes in the ventral ectoderm also involves both the AB region and the Eh-1 motif; FoxD4/5 accomplishes ectopic neural induction by both activating neural precursor genes and repressing BMP signaling and epidermal genes. This study identifies the specific, conserved domains of the FoxD4/5 protein that allow this single transcription factor to regulate a network of genes that controls the transition of a proliferative neural ectodermal population to a committed neural plate population poised to begin differentiation.

  17. Role of Flightless-I (Drosophila) homolog in the transcription activation of type I collagen gene mediated by transforming growth factor beta

    SciTech Connect

    Lim, Mi-Sun; Jeong, Kwang Won

    2014-11-21

    Highlights: • FLII activates TGFβ-mediated expression of COL1A2 gene. • TGFβ induces the association of FLII with SMAD3 and BRG1 in A549 cells. • FLII is required for the recruitment of SWI/SNF complex and chromatin accessibility to COL1A2 promoter. - Abstract: Flightless-I (Drosophila) homolog (FLII) is a nuclear receptor coactivator that is known to interact with other transcriptional regulators such as the SWI/SNF complex, an ATP-dependent chromatin-remodeling complex, at the promoter or enhancer region of estrogen receptor (ER)-α target genes. However, little is known about the role of FLII during transcription initiation in the transforming growth factor beta (TGFβ)/SMAD-dependent signaling pathway. Here, we demonstrate that FLII functions as a coactivator in the expression of type I collagen gene induced by TGFβ in A549 cells. FLII activates the reporter gene driven by COL1A2 promoter in a dose-dependent manner. Co-expression of GRIP1, CARM1, or p300 did not show any synergistic activation of transcription. Furthermore, the level of COL1A2 expression correlated with the endogenous level of FLII mRNA level. Depletion of FLII resulted in a reduction of TGFβ-induced expression of COL1A2 gene. In contrast, over-expression of FLII caused an increase in the endogenous expression of COL1A2. We also showed that FLII is associated with Brahma-related gene 1 (BRG1) as well as SMAD in A549 cells. Notably, the recruitment of BRG1 to the COL1A2 promoter region was decreased in FLII-depleted A549 cells, suggesting that FLII is required for TGFβ-induced chromatin remodeling, which is carried out by the SWI/SNF complex. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments revealed that depletion of FLII caused a reduction in chromatin accessibility at the COL1A2 promoter. These results suggest that FLII plays a critical role in TGFβ/SMAD-mediated transcription of the COL1A2 gene

  18. Functional analysis of differences in transcriptional activity conferred by genetic variants in the 5' flanking region of the IL12RB2 gene.

    PubMed

    Kato-Kogoe, Nahoko; Ohyama, Hideki; Okano, Soichiro; Yamanegi, Koji; Yamada, Naoko; Hata, Masaki; Nishiura, Hiroshi; Abiko, Yoshimitsu; Terada, Nobuyuki; Nakasho, Keiji

    2016-01-01

    Interleukin 12 receptor β chain (IL12RB2) is a crucial regulatory factor involved in cell-mediated immune responses, and genetic variants of the gene encoding IL12RB2 are associated with susceptibility to various immune-related diseases. We previously demonstrated that haplotypes with single nucleotide polymorphisms (SNPs) in the 5' flanking region of IL12RB2, including -1035A>G (rs3762315) and -1023A>G (rs3762316), affect the expression of IL12RB2, thereby altering susceptibility to leprosy and periodontal diseases. In the present study, we identified transcription factors associated with the haplotype-specific transcriptional activity of IL12RB2 in T cells and NK cells. The -1023G polymorphism was found to create a consensus binding site for the transcription factor activating protein (AP)-1, and enzyme-linked immunosorbent assay (ELISA)-based binding assays showed that these SNPs enhanced AP-1 binding to this region. In reporter assays, suppression of JunB expression using siRNA eliminated differences in the -1035G/-1023G and -1035A/-1023A regions containing IL12RB2 promoter activity in Jurkat T cells and NK3.3 cells. These results suggested that the -1035/-1023 polymorphisms created differential binding affinities for JunB that could lead to differential IL12RB2 expression. Moreover, the -1035G and -1035A alleles formed binding sites for GATA-3 and myocyte enhancer factor-2 (MEF-2), respectively. Our data indicated that in addition to JunB, the SNP at -1035/-1023 influenced GATA-3 and MEF-2 binding affinity, potentially altering IL12RB2 transcriptional activity. These findings confirm the effects of rs3762315 and rs3762316 on IL12RB2 transcription. These genetic variants may alter cellular activation of T cells and NK cells and modify cell-mediated immune responses.

  19. Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-κB target genes in human breast cancer

    PubMed Central

    Dalmases, Alba; González, Irene; Menendez, Silvia; Arpí, Oriol; Corominas, Josep Maria; Servitja, Sonia; Tusquets, Ignasi; Chamizo, Cristina; Rincón, Raúl; Espinosa, Lluis; Bigas, Anna; Eroles, Pilar; Furriol, Jessica; Lluch, Anna; Rovira, Ana; Albanell, Joan; Rojo, Federico

    2014-01-01

    NF-κB has been linked to doxorubicin resistance in breast cancer patients. NF-κB nuclear translocation and DNA binding in doxorubicin treated-breast cancer cells have been extensively examined; however its functional relevance at transcriptional level on NF-κB -dependent genes and the biological consequences are unclear. We studied NF-κB -dependent gene expression induced by doxorubicin in breast cancer cells and fresh human cancer specimens with different genetic backgrounds focusing on their p53 status. NF-κB -dependent signature of doxorubicin was identified by gene expression microarrays in breast cancer cells treated with doxorubicin and the IKKβ-inhibitor MLN120B, and confirmed ex vivo in human cancer samples. The association with p53 was functionally validated. Finally, NF-κB activation and p53 status was determined in a cohort of breast cancer patients treated with adjuvant doxorubicin-based chemotherapy. Doxorubicin treatment in the p53-mutated MDA-MB-231 cells resulted in NF NF-κB driven-gene transcription signature. Modulation of genes related with invasion, metastasis and chemoresistance (ICAM-1, CXCL1, TNFAIP3, IL8) were confirmed in additional doxorubicin-treated cell lines and fresh primary human breast tumors. In both systems, p53-defcient background correlated with the activation of the NF-κB -dependent signature. Furthermore, restoration of p53WT in the mutant p53 MDA-MB-231 cells impaired NF-κB driven transcription induced by doxorubicin. Moreover, a p53 deficient background and nuclear NF-κB /p65 in breast cancer patients correlated with reduced disease free-survival. This study supports that p53 deficiency is necessary for a doxorubicin driven NF-κB -response that limits doxorubicin cytotoxicity in breast cancer and is linked to an aggressive clinical behavior. PMID:24344116

  20. Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-кB target genes in human breast cancer.

    PubMed

    Dalmases, Alba; González, Irene; Menendez, Silvia; Arpí, Oriol; Corominas, Josep Maria; Servitja, Sonia; Tusquets, Ignasi; Chamizo, Cristina; Rincón, Raúl; Espinosa, Lluis; Bigas, Anna; Eroles, Pilar; Furriol, Jessica; Lluch, Anna; Rovira, Ana; Albanell, Joan; Rojo, Federico

    2014-01-15

    NF-кB has been linked to doxorubicin resistance in breast cancer patients. NF-кB nuclear translocation and DNA binding in doxorubicin treated-breast cancer cells have been extensively examined; however its functional relevance at transcriptional level on NF-кB-dependent genes and the biological consequences are unclear. We studied NF-кB-dependent gene expression induced by doxorubicin in breast cancer cells and fresh human cancer specimens with different genetic backgrounds focusing on their p53 status. NF-кB-dependent signature of doxorubicin was identified by gene expression microarrays in breast cancer cells treated with doxorubicin and the IKKβ-inhibitor MLN120B, and confirmed ex vivo in human cancer samples. The association with p53 was functionally validated. Finally, NF-кB activation and p53 status was determined in a cohort of breast cancer patients treated with adjuvant doxorubicin-based chemotherapy. Doxorubicin treatment in the p53-mutated MDA-MB-231 cells resulted in NF-кB driven-gene transcription signature. Modulation of genes related with invasion, metastasis and chemoresistance (ICAM-1, CXCL1, TNFAIP3, IL8) were confirmed in additional doxorubicin-treated cell lines and fresh primary human breast tumors. In both systems, p53-deficient background correlated with the activation of the NF-кB-dependent signature. Furthermore, restoration of p53WT in the mutant p53 MDA-MB-231 cells impaired NF-кB driven transcription induced by doxorubicin. Moreover, a p53 deficient background and nuclear NF-кB/p65 in breast cancer patients correlated with reduced disease free-survival. This study supports that p53 deficiency is necessary for a doxorubicin driven NF-кB-response that limits doxorubicin cytotoxicity in breast cancer and is linked to an aggressive clinical behavior.

  1. Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships

    PubMed Central

    Booher, Nicholas J.; Carpenter, Sara C. D.; Sebra, Robert P.; Wang, Li; Salzberg, Steven L.; Leach, Jan E.; Bogdanove, Adam J.

    2016-01-01

    Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33–35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution. PMID:27148456

  2. Linking Transcriptional Changes over Time in Stimulated Dendritic Cells to Identify Gene Networks Activated during the Innate Immune Response

    PubMed Central

    Patil, Ashwini; Kumagai, Yutaro; Liang, Kuo-ching; Suzuki, Yutaka; Nakai, Kenta

    2013-01-01

    The innate immune response is primarily mediated by the Toll-like receptors functioning through the MyD88-dependent and TRIF-dependent pathways. Despite being widely studied, it is not yet completely understood and systems-level analyses have been lacking. In this study, we identified a high-probability network of genes activated during the innate immune response using a novel approach to analyze time-course gene expression profiles of activated immune cells in combination with a large gene regulatory and protein-protein interaction network. We classified the immune response into three consecutive time-dependent stages and identified the most probable paths between genes showing a significant change in expression at each stage. The resultant network contained several novel and known regulators of the innate immune response, many of which did not show any observable change in expression at the sampled time points. The response network shows the dominance of genes from specific functional classes during different stages of the immune response. It also suggests a role for the protein phosphatase 2a catalytic subunit α in the regulation of the immunoproteasome during the late phase of the response. In order to clarify the differences between the MyD88-dependent and TRIF-dependent pathways in the innate immune response, time-course gene expression profiles from MyD88-knockout and TRIF-knockout dendritic cells were analyzed. Their response networks suggest the dominance of the MyD88-dependent pathway in the innate immune response, and an association of the circadian regulators and immunoproteasomal degradation with the TRIF-dependent pathway. The response network presented here provides the most probable associations between genes expressed in the early and the late phases of the innate immune response, while taking into account the intermediate regulators. We propose that the method described here can also be used in the identification of time-dependent gene sub

  3. Linking transcriptional changes over time in stimulated dendritic cells to identify gene networks activated during the innate immune response.

    PubMed

    Patil, Ashwini; Kumagai, Yutaro; Liang, Kuo-Ching; Suzuki, Yutaka; Nakai, Kenta

    2013-01-01

    The innate immune response is primarily mediated by the Toll-like receptors functioning through the MyD88-dependent and TRIF-dependent pathways. Despite being widely studied, it is not yet completely understood and systems-level analyses have been lacking. In this study, we identified a high-probability network of genes activated during the innate immune response using a novel approach to analyze time-course gene expression profiles of activated immune cells in combination with a large gene regulatory and protein-protein interaction network. We classified the immune response into three consecutive time-dependent stages and identified the most probable paths between genes showing a significant change in expression at each stage. The resultant network contained several novel and known regulators of the innate immune response, many of which did not show any observable change in expression at the sampled time points. The response network shows the dominance of genes from specific functional classes during different stages of the immune response. It also suggests a role for the protein phosphatase 2a catalytic subunit α in the regulation of the immunoproteasome during the late phase of the response. In order to clarify the differences between the MyD88-dependent and TRIF-dependent pathways in the innate immune response, time-course gene expression profiles from MyD88-knockout and TRIF-knockout dendritic cells were analyzed. Their response networks suggest the dominance of the MyD88-dependent pathway in the innate immune response, and an association of the circadian regulators and immunoproteasomal degradation with the TRIF-dependent pathway. The response network presented here provides the most probable associations between genes expressed in the early and the late phases of the innate immune response, while taking into account the intermediate regulators. We propose that the method described here can also be used in the identification of time-dependent gene sub

  4. Characteristics of post-transcriptional gene silencing.

    PubMed

    Chicas, A; Macino, G

    2001-11-01

    A number of gene silencing phenomena that inactivate genes at the post-transcriptional level have been identified. Due to its potential for studying gene function, post-transcriptional gene silencing (PTGS) has become an intense area of research. In this review we describe the different means of inducing PTGS and discuss the possible biological roles of these artificially induced phenomena. We also discuss other features of PTGS such as the mechanism of mRNA degradation, the nature of the silencing signal and the mechanism of PTGS inhibition by viral proteins.

  5. Characteristics of post-transcriptional gene silencing

    PubMed Central

    Chicas, Agustin; Macino, Giuseppe

    2001-01-01

    A number of gene silencing phenomena that inactivate genes at the post-transcriptional level have been identified. Due to its potential for studying gene function, post-transcriptional gene silencing (PTGS) has become an intense area of research. In this review we describe the different means of inducing PTGS and discuss the possible biological roles of these artificially induced phenomena. We also discuss other features of PTGS such as the mechanism of mRNA degradation, the nature of the silencing signal and the mechanism of PTGS inhibition by viral proteins. PMID:11713190

  6. Combining modelling and experimental approaches to explain how calcium signatures are decoded by calmodulin-binding transcription activators (CAMTAs) to produce specific gene expression responses.

    PubMed

    Liu, Junli; Whalley, Helen J; Knight, Marc R

    2015-10-01

    Experimental data show that Arabidopsis thaliana is able to decode different calcium signatures to produce specific gene expression responses. It is also known that calmodulin-binding transcription activators (CAMTAs) have calmodulin (CaM)-binding domains. Therefore, the gene expression responses regulated by CAMTAs respond to calcium signals. However, little is known about how different calcium signatures are decoded by CAMTAs to produce specific gene expression responses. A dynamic model of Ca(2+) -CaM-CAMTA binding and gene expression responses is developed following thermodynamic and kinetic principles. The model is parameterized using experimental data. Then it is used to analyse how different calcium signatures are decoded by CAMTAs to produce specific gene expression responses. Modelling analysis reveals that: calcium signals in the form of cytosolic calcium concentration elevations are nonlinearly amplified by binding of Ca(2+) , CaM and CAMTAs; amplification of Ca(2+) signals enables calcium signatures to be decoded to give specific CAMTA-regulated gene expression responses; gene expression responses to a calcium signature depend upon its history and accumulate all the information during the lifetime of the calcium signature. Information flow from calcium signatures to CAMTA-regulated gene expression responses has been established by combining experimental data with mathematical modelling.

  7. Modulation of Elk-dependent-transcription by Gene33.

    PubMed

    Keeton, Adam B; Messina, Joseph L

    2005-04-15

    Gene33 is a cytoplasmic protein expressed in many cell types, including those of renal and hepatic origin. Its expression is regulated by a large number of mitogenic and stressful stimuli, both in cultured cells and in vivo. Gene33 protein possesses binding domains for ErbB receptors, 14-3-3 proteins, SH-3 domains, and GTP bound Cdc42, suggesting that it may play a role in signal transduction. Indeed, these regions of Gene33 have been reported to modulate signaling through the ERK, JNK, and NFkappaB pathways. In the present work, epitope-tagged full-length and truncation mutants, as well as wild-type Gene33, were overexpressed in 293 cells. The expression of these proteins was compared to the level of endogenous Gene33 by Western blot using a newly developed polyclonal antibody. As proxies for activity of the ERK and JNK pathways, Elk- and c-Jun-dependent transcription were measured by a luciferase reporter gene. Moderate expression levels of full-length Gene33 caused a twofold increase in Elk-dependent transcription, while at higher levels, c-Jun-dependent transcription was partially inhibited. The C-terminal half of Gene33 significantly increased both Elk- and c-Jun-dependent transcription when expressed at approximately threefold above control levels. This effect on Elk-dependent transcription was lost at higher levels of Gene33 expression. In contrast, higher levels of the C-terminal half of Gene33 caused a progressively greater effect on c-Jun-dependent transcription. These findings suggest that Gene33 may increase ERK activity, and that the C-terminal half of Gene33 may act less specifically in the absence of the N-terminal half, inducing JNK activity.

  8. Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

    PubMed

    Min, Jiho; Jin, Yoon-Mi; Moon, Je-Sung; Sung, Min-Sun; Jo, Sangmee Ahn; Jo, Inho

    2006-06-01

    Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.

  9. Knockout of the adp gene related with colonization in Bacillus nematocida B16 using customized transcription activator-like effectors nucleases

    PubMed Central

    Niu, Qiuhong; Zheng, Haoying; Zhang, Lin; Qin, Fujun; Facemire, Loryn; Zhang, Guo; Cao, Feng; Zhang, Ke-qin; Huang, Xiaowei; Yang, Jianwei; He, Lei; Liu, Chanjuan

    2015-01-01

    Bacillus nematocida B16 is able to dominate in the intestines of the worm Caenorhabditis elegans in ‘Trojan horse’ pathogenic mechanism. The adp is one candidate gene which potentially play a vital role in the colonization from our previous random mutagenesis screening results. To analyse the functional role of this gene, we constructed the adp knockout mutant through customized transcription activator-like effectors nucleases (TALEN), which has been successfully used in yeasts, nematodes, zebrafish and human pluripotent cells. Here, we first time report this knockout method in bacteria on this paper. Bioassay experiments demonstrated that the adp knockout mutant of B16 showed considerably lower colonization activity, reduced numbers of intestines and less than 80% nematocidal activity compared with the wild-type strain when infected for 48 h. However, no obvious change on proteolytic activity was observed in the mutant. Conversely, the complementation of adp gene restored most of the above deficient phenotypes. These results indicated that the adp gene was involved in surface adhesion and played a comparatively important role in colonizing host nematodes. Moreover, TALENs successfully disrupt target genes in bacteria. PMID:25912819

  10. Transcriptional regulation by activation and repression elements located at the 5'-noncoding region of the human alpha9 nicotinic receptor subunit gene.

    PubMed

    Valor, Luis M; Castillo, Mar; Ortiz, José A; Criado, Manuel

    2003-09-26

    The alpha9 subunit is a component of the neuronal nicotinic acetylcholine receptor gene superfamily that is expressed in very restricted locations. The promoter of the human gene has been analyzed in the human neuroblastoma SH-SY5Y, where alpha9 subunit expression was detected, and in C2C12 cells that do not express alpha9. A proximal promoter region (from -322 to +113) showed maximal transcriptional activity in SH-SY5Y cells, whereas its activity in C1C12 cells was much lower. Two elements unusually located at the 5'-noncoding region exhibited opposite roles. A negative element located between +15 and +48 appears to be cell-specific because it was effective in C2C12 but not in SH-SY5Y cells, where it was counterbalanced by the presence of the promoter region 5' to the initiation site. An activating element located between +66 and +79 and formed by two adjacent Sox boxes increased the activity of the alpha9 promoter about 4-fold and was even able to activate other promoters. This element interacts with Sox proteins, probably through a cooperative mechanism in which the two Sox boxes are necessary. We propose that the Sox complex provides an initial scaffold that facilitates the recruiting of the transcriptional machinery responsible for alpha9 subunit expression.

  11. The external amino acid signaling pathway promotes activation of Stp1 and Uga35/Dal81 transcription factors for induction of the AGP1 gene in Saccharomyces cerevisiae.

    PubMed Central

    Abdel-Sater, Fadi; Iraqui, Ismaïl; Urrestarazu, Antonio; André, Bruno

    2004-01-01

    Yeast cells respond to the presence of amino acids in their environment by inducing transcription of several amino acid permease genes including AGP1, BAP2, and BAP3. The signaling pathway responsible for this induction involves Ssy1, a permease-like sensor of external amino acids, and culminates with proteolytic cleavage and translocation to the nucleus of the zinc-finger proteins Stp1 and Stp2, the lack of which abolishes induction of BAP2 and BAP3. Here we show that Stp1-but not Stp2-plays an important role in AGP1 induction, although significant induction of AGP1 by amino acids persists in stp1 and stp1 stp2 mutants. This residual induction depends on the Uga35/Dal81 transcription factor, indicating that the external amino acid signaling pathway activates not only Stp1 and Stp2, but also another Uga35/Dal81-dependent transcriptional circuit. Analysis of the AGP1 gene's upstream region revealed that Stp1 and Uga35/Dal81 act synergistically through a 21-bp cis-acting sequence similar to the UAS(AA) element previously found in the BAP2 and BAP3 upstream regions. Although cells growing under poor nitrogen-supply conditions display much higher induction of AGP1 expression than cells growing under good nitrogen-supply conditions, the UAS(AA) itself is totally insensitive to nitrogen availability. Nitrogen-source control of AGP1 induction is mediated by the GATA factor Gln3, likely acting through adjacent 5'-GATA-3' sequences, to amplify the positive effect of UAS(AA). Our data indicate that Stp1 may act in combination with distinct sets of transcription factors, according to the gene context, to promote induction of transcription in response to external amino acids. The data also suggest that Uga35/Dal81 is yet another transcription factor under the control of the external amino acid sensing pathway. Finally, the data show that the TOR pathway mediating global nitrogen control of transcription does not interfere with the external amino acid signaling pathway. PMID

  12. Physical separation of two different forms of human TFIIIB active in the transcription of the U6 or the VAI gene in vitro.

    PubMed

    Teichmann, M; Seifart, K H

    1995-12-01

    Human transcription factor hTFIIIB is necessary to initiate transcription correctly from all RNA polymerase III (pol III) genes which are governed by structurally different promoters, and it is unclear whether hTFIIIB complexes, required for intragenic or 5'-located pol III promoters, are composed of unique or different components. We show here that two different forms of hTFIIIB can be separated physically by ion exchange chromatography. hTFIIIB-alpha shows strong preference for transcription of the U6 over the VAI gene and does not contain TATA binding protein (TBP). After SDS-PAGE and renaturation of proteins, the transcriptional activity of hTFIIIB-alpha can be reconstituted by fractions corresponding to a mean M(r) of 25, 60 and 90 kDa. Upon gradient centrifugation or gel filtration, the activity of hTFIIIB-alpha is associated with an M(r) of 60 +/- 10 kDa, indicating that the components of the complex tend to dissociate. In contrast, hTFIIIB-beta is predominantly active on intragenic pol III promoters. It reveals an M(r) of 300 +/- 30 kDa upon gel filtration and, besides TBP, it contains several associated factors (TAFs). Two of these proteins reveal an M(r) of 60 kDa and 90 kDa, and it is conceivable that they are related to polypeptides of similar mass functionally identified in hTFIIIB-alpha. These protein are probably required for the recruitment of pol III to the initiation site at 5'-located and intragenic promoters.

  13. Isolation and expression profile of a gene encoding for the Signal Transducer and Activator of Transcription STAT2 in Atlantic salmon (Salmo salar).

    PubMed

    Collet, Bertrand; Ganne, Géraldine; Bird, Steve; Collins, Catherine M

    2009-07-01

    Signal Transducer and Activator of Transcription (STAT)-2 is a molecule involved in the type I interferon (IFN) signalling pathway. The full length cDNA sequence of Atlantic salmon (Salmo salar) ssSTAT2 was determined and phylogenetic analysis of the amino acid sequence grouped this novel salmon gene to the STAT2 clade. This represents the first fish STAT2 report. The gene encodes for a 802 aa polypeptide that has 38% identity to the human or murine STAT2. The expression was monitored by qPCR in the kidney of animals over the time of infection with the Infectious Salmon Anaemia Virus (ISAV) and in TO cells infected with Infectious Pancreatic Necrosis Virus (IPNV) or with the Salmon Alphavirus (SAV). SAV and ISAV induced an approximate 10-fold increase in the level of expression of ssSTAT2 gene whilst IPNV only induced a 1.5-fold increase.

  14. Reverse engineering transcriptional gene networks.

    PubMed

    Belcastro, Vincenzo; di Bernardo, Diego

    2014-01-01

    The aim of this chapter is a step-by-step guide on how to infer gene networks from gene expression profiles. The definition of a gene network is given in Subheading 1, where the different types of networks are discussed. The chapter then guides the readers through a data-gathering process in order to build a compendium of gene expression profiles from a public repository. Gene expression profiles are then discretized and a statistical relationship between genes, called mutual information (MI), is computed. Gene pairs with insignificant MI scores are then discarded by applying one of the described pruning steps. The retained relationships are then used to build up a Boolean adjacency matrix used as input for a clustering algorithm to divide the network into modules (or communities). The gene network can then be used as a hypothesis generator for discovering gene function and analyzing gene signatures. Some case studies are presented, and an online web-tool called Netview is described.

  15. Effects of zinc on CarE activities and its gene transcript level in the English grain aphid, Sitobion avenae.

    PubMed

    Gao, Huan-Huan; Zhao, Hui-Yan; Yang, Jie; Zhang, Li; Bai, Xiao-Hui; Hu, Zu-Qing; Hu, Xiang-Shun

    2014-05-15

    As a selective stress, heavy metals play an important role in inducing the adaptive adjustments of insects to changing environments. Carboxylesterase (CarE) is one kind of biomarker that could help us to explore the adaptation mechanism of aphids to heavy metal stress. In this study, CarE activity and gene expression level were investigated in English grain aphids, Sitobion avenae (F.) (Hemiptera: Aphididae), exposed to Zn2+ at concentrations of 0, 400, and 1600 mg/kg for 5, 15, 25, and 30 generations. The results showed that the CarE activity was significantly different between different Zn2+ concentrations and different generations. The CarE activity significantly decreased with increasing generations. In the higher generations, the CarE activity was strongly inhibited by the 1600 mg/kg of Zn2+. Realtime quantitative PCR revealed that the CarE gene expression pattern in S. avenae was up-regulated under the condition of 400 mg/kg and 1600 mg/kg of Zn2+, but a significant difference was not found after long-term exposure to high concentrations of Zn2+. It was concluded that CarE could be the sensitive biomarker for S. avenae response to the presence of Zn2+. In order to adapt to heavy metal Zn2+ stress, S. avenae had particular patterns of gene expression under long-term stress.

  16. Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1.

    PubMed Central

    Baler, R; Dahl, G; Voellmy, R

    1993-01-01

    Transcriptional activity of heat shock (hsp) genes is controlled by a heat-activated, group-specific transcription factor(s) recognizing arrays of inverted repeats of the element NGAAN. To date genes for two human factors, HSF1 and HSF2, have been isolated. To define their properties as well as the changes they undergo during heat stress activation, we prepared polyclonal antibodies to these factors. Using these tools, we have shown that human HeLa cells constitutively synthesize HSF1, but we were unable to detect HSF2. In unstressed cells HSF1 is present mainly in complexes with an apparent molecular mass of about 200 kDa, unable to bind to DNA. Heat treatment induces a shift in the apparent molecular mass of HSF1 to about 700 kDa, concomitant with the acquisition of DNA-binding ability. Cross-linking experiments suggest that this change in complex size may reflect the trimerization of monomeric HSF1. Human HSF1 expressed in Xenopus oocytes does not bind DNA, but derepression of DNA-binding activity, as well as oligomerization of HSF1, occurs during heat treatment at the same temperature at which hsp gene expression is induced in this organism, suggesting that a conserved Xenopus protein(s) plays a role in this regulation. Inactive HSF1 resides in the cytoplasm of human cells; on activation it rapidly translocates to a soluble nuclear fraction, and shortly thereafter it becomes associated with the nuclear pellet. On heat shock, activatable HSF1, which might already have been posttranslationally modified in the unstressed cell, undergoes further modification. These different process provide multiple points of regulation of hsp gene expression. Images PMID:8455624

  17. The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts.

    PubMed

    Heras, Sara R; López, Manuel C; Olivares, Mónica; Thomas, M Carmen

    2007-01-01

    L1Tc is the best represented autonomous LINE of the Trypanosoma cruzi genome, throughout which several functional copies may exist. In this study, we show that the first 77 bp of L1Tc (Pr77) (also present in the T. cruzi non-autonomous retrotransposon NARTc, in the Trypanosoma brucei RIME/ingi elements, and in the T. cruzi, T. brucei and Leishmania major degenerate L1Tc/ingi-related elements [DIREs]) behave as a promoter element that activates gene transcription. The transcription rate promoted by Pr77 is 10-14-fold higher than that mediated by sequences located upstream from the T. cruzi tandemly repeated genes KMP11 and the GAPDH. The Pr77 promoter-derived mRNAs initiate at nucleotide +1 of L1Tc, are unspliced and translated. L1Tc transcripts show a moderate half life and are RNA pol II dependent. The presence of an internal promoter at the 5' end of L1Tc favors the production of full-length L1Tc RNAs and reinforces the hypothesis that this mobile element may be naturally autonomous in its transposition.

  18. Androgen receptor stimulates bone sialoprotein (BSP) gene transcription via cAMP response element and activator protein 1/glucocorticoid response elements.

    PubMed

    Takai, Hideki; Nakayama, Youhei; Kim, Dong-Soon; Arai, Masato; Araki, Shouta; Mezawa, Masaru; Nakajima, Yu; Kato, Naoko; Masunaga, Hiroshi; Ogata, Yorimasa

    2007-09-01

    Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. Androgens are steroid hormones that are essential for skeletal development. The androgen receptor (AR) is a transcription factor and a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. To determine the molecular mechanism involved in the stimulation of bone formation, we have analyzed the effects of androgens and AR effects on BSP gene transcription. AR protein levels were increased after AR overexpression in ROS17/2.8 cells. BSP mRNA levels were increased by AR overexpression. However, the endogenous and overexpressed BSP mRNA levels were not changed by DHT (10(-8) M, 24 h). Whereas luciferase (LUC) activities in all constructs, including a short construct (nts -116 to +60), were increased by AR overexpression, the basal and LUC activities enhanced by AR overexpression were not induced by DHT (10(-8)M, 24 h). The effect of AR overexpression was abrogated by 2 bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that AR overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE-protein complexes were supershifted by phospho-CREB antibody, and CREB, c-Fos, c-Jun, and AR antibodies disrupted the complexes formation. The AP1/GRE-protein complexes were supershifted by c-Fos antibody and c-Jun, and AR antibodies disrupted the complexes formation. These studies demonstrate that AR stimulates BSP gene transcription by targeting the CRE and AP1/GRE elements in the promoter of the rat BSP gene.

  19. Transcriptional Activity of the MADS Box ARLEQUIN/TOMATO AGAMOUS-LIKE1 Gene Is Required for Cuticle Development of Tomato Fruit1

    PubMed Central

    Giménez, Estela; Dominguez, Eva; Pineda, Benito; Heredia, Antonio; Moreno, Vicente; Angosto, Trinidad

    2015-01-01

    Fruit development and ripening entail key biological and agronomic events, which ensure the appropriate formation and dispersal of seeds and determine productivity and yield quality traits. The MADS box gene ARLEQUIN/TOMATO AGAMOUS-LIKE1 (hereafter referred to as TAGL1) was reported as a key regulator of tomato (Solanum lycopersicum) reproductive development, mainly involved in flower development, early fruit development, and ripening. It is shown here that silencing of the TAGL1 gene (RNA interference lines) promotes significant changes affecting cuticle development, mainly a reduction of thickness and stiffness, as well as a significant decrease in the content of cuticle components (cutin, waxes, polysaccharides, and phenolic compounds). Accordingly, overexpression of TAGL1 significantly increased the amount of cuticle and most of its components while rendering a mechanically weak cuticle. Expression of the genes involved in cuticle biosynthesis agreed with the biochemical and biomechanical features of cuticles isolated from transgenic fruits; it also indicated that TAGL1 participates in the transcriptional control of cuticle development mediating the biosynthesis of cuticle components. Furthermore, cell morphology and the arrangement of epidermal cell layers, on whose activity cuticle formation depends, were altered when TAGL1 was either silenced or constitutively expressed, indicating that this transcription factor regulates cuticle development, probably through the biosynthetic activity of epidermal cells. Our results also support cuticle development as an integrated event in the fruit expansion and ripening processes that characterize fleshy-fruited species such as tomato. PMID:26019301

  20. Transcriptional Activity of the MADS Box ARLEQUIN/TOMATO AGAMOUS-LIKE1 Gene Is Required for Cuticle Development of Tomato Fruit.

    PubMed

    Giménez, Estela; Dominguez, Eva; Pineda, Benito; Heredia, Antonio; Moreno, Vicente; Lozano, Rafael; Angosto, Trinidad

    2015-07-01

    Fruit development and ripening entail key biological and agronomic events, which ensure the appropriate formation and dispersal of seeds and determine productivity and yield quality traits. The MADS box gene Arlequin/tomato Agamous-like1 (hereafter referred to as TAGL1) was reported as a key regulator of tomato (Solanum lycopersicum) reproductive development, mainly involved in flower development, early fruit development, and ripening. It is shown here that silencing of the TAGL1 gene (RNA interference lines) promotes significant changes affecting cuticle development, mainly a reduction of thickness and stiffness, as well as a significant decrease in the content of cuticle components (cutin, waxes, polysaccharides, and phenolic compounds). Accordingly, overexpression of TAGL1 significantly increased the amount of cuticle and most of its components while rendering a mechanically weak cuticle. Expression of the genes involved in cuticle biosynthesis agreed with the biochemical and biomechanical features of cuticles isolated from transgenic fruits; it also indicated that TAGL1 participates in the transcriptional control of cuticle development mediating the biosynthesis of cuticle components. Furthermore, cell morphology and the arrangement of epidermal cell layers, on whose activity cuticle formation depends, were altered when TAGL1 was either silenced or constitutively expressed, indicating that this transcription factor regulates cuticle development, probably through the biosynthetic activity of epidermal cells. Our results also support cuticle development as an integrated event in the fruit expansion and ripening processes that characterize fleshy-fruited species such as tomato.

  1. Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts

    NASA Technical Reports Server (NTRS)

    Thomas, M. J.; Umayahara, Y.; Shu, H.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1996-01-01

    Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal

  2. Convergent Transcription of Interferon-stimulated Genes by TNF-α and IFN-α Augments Antiviral Activity against HCV and HEV.

    PubMed

    Wang, Wenshi; Xu, Lei; Brandsma, Johannes H; Wang, Yijin; Hakim, Mohamad S; Zhou, Xinying; Yin, Yuebang; Fuhler, Gwenny M; van der Laan, Luc J W; van der Woude, C Janneke; Sprengers, Dave; Metselaar, Herold J; Smits, Ron; Poot, Raymond A; Peppelenbosch, Maikel P; Pan, Qiuwei

    2016-05-06

    IFN-α has been used for decades to treat chronic hepatitis B and C, and as an off-label treatment for some cases of hepatitis E virus (HEV) infection. TNF-α is another important cytokine involved in inflammatory disease, which can interact with interferon signaling. Because interferon-stimulated genes (ISGs) are the ultimate antiviral effectors of the interferon signaling, this study aimed to understand the regulation of ISG transcription and the antiviral activity by IFN-α and TNF-α. In this study, treatment of TNF-α inhibited replication of HCV by 71 ± 2.4% and HEV by 41 ± 4.9%. Interestingly, TNF-α induced the expression of a panel of antiviral ISGs (2-11 fold). Blocking the TNF-α signaling by Humira abrogated ISG induction and its antiviral activity. Chip-seq data analysis and mutagenesis assay further revealed that the NF-κB protein complex, a key downstream element of TNF-α signaling, directly binds to the ISRE motif in the ISG promoters and thereby drives their transcription. This process is independent of interferons and JAK-STAT cascade. Importantly, when combined with IFN-α, TNF-α works cooperatively on ISG induction, explaining their additive antiviral effects. Thus, our study reveals a novel mechanism of convergent transcription of ISGs by TNF-α and IFN-α, which augments their antiviral activity against HCV and HEV.

  3. Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

    PubMed Central

    Lee, Bomi; Wu, Cheng-Ying; Lin, Yi-Wei; Park, Sung Wook; Wei, Li-Na

    2016-01-01

    All-trans Retinoic acid (RA) and its derivatives are potent therapeutics for immunological functions including wound repair. However, the molecular mechanism of RA modulation in innate immunity is poorly understood, especially in macrophages. We found that topical application of RA significantly improves wound healing and that RA and IL-4 synergistically activate Arg1, a critical gene for tissue repair, in M2 polarized macrophages. This involves feed forward regulation of Raldh2, a rate-limiting enzyme for RA biosynthesis, and requires Med25 to coordinate RAR, STAT6 and chromatin remodeler, Brg1 to remodel the +1 nucleosome of Arg1 for transcription initiation. By recruiting elongation factor TFIIS, Med25 also facilitates transcriptional initiation-elongation coupling. This study uncovers synergistic activation of Arg1 by RA and IL-4 in M2 macrophages that involves feed forward regulation of RA synthesis and dual functions of Med25 in nucleosome remodeling and transcription initiation-elongation coupling that underlies robust modulatory activity of RA in innate immunity. PMID:27166374

  4. Post-transcriptional gene silencing suppressor activity of two non-pathogenic alphasatellites associated with a begomovirus.

    PubMed

    Nawaz-Ul-Rehman, Muhammad Shah; Nahid, Nazia; Mansoor, Shahid; Briddon, Rob W; Fauquet, Claude M

    2010-09-30

    Alphasatellites and betasatellites are begomovirus-associated single-stranded circular DNA molecules. Two distinct alphasatellites, Gossypium darwinii symptomless alphasatellite and Gossypium mustelinium symptomless alphasatellite, were previously isolated from Gossypium davidsonii and G.mustelinium. Here we show that the replication-associated proteins (Rep: a rolling-circle replication initiator protein) encoded by these alphasatellites interact with the Rep and C4 proteins encoded by their helper begomovirus, Cotton leaf curl Rajasthan virus (CLCuRaV), in a yeast two-hybrid assay. Both the alphasatellite-encoded Reps were found to have strong gene silencing suppressor activity, in contrast to the betasatellite-encoded betaC1 and CLCuRaV-encoded C2, C4 and V2 proteins. The presence of alphasatellites maintained suppression of gene silencing in the youngest, actively growing tissue of CLCuRaV-betasatellite-infected plants. This is the first demonstration of a rolling-circle replication initiator protein with suppressor of gene silencing activity and provides a possible explanation for the selective advantage provided by the association of alphasatellites with begomovirus-betasatellite complexes.

  5. Transcription activation by the adenovirus E1a protein

    NASA Astrophysics Data System (ADS)

    Lillie, James W.; Green, Michael R.

    1989-03-01

    The adenovirus Ela protein stimulates transcription of a wide variety of viral and cellular genes. It is shown here that Ela has the two functions characteristic of a typical cellular activator: one direct Ela to the promoter, perhaps by interacting with a DMA-bound protein, and the other, an activating region, enables the bound activator to stimulate transcription.

  6. Regulation of Lactobacillus casei Sorbitol Utilization Genes Requires DNA-Binding Transcriptional Activator GutR and the Conserved Protein GutM▿

    PubMed Central

    Alcántara, Cristina; Sarmiento-Rubiano, Luz Adriana; Monedero, Vicente; Deutscher, Josef; Pérez-Martínez, Gaspar; Yebra, María J.

    2008-01-01

    Sequence analysis of the five genes (gutRMCBA) downstream from the previously described sorbitol-6-phosphate dehydrogenase-encoding Lactobacillus casei gutF gene revealed that they constitute a sorbitol (glucitol) utilization operon. The gutRM genes encode putative regulators, while the gutCBA genes encode the EIIC, EIIBC, and EIIA proteins of a phosphoenolpyruvate-dependent sorbitol phosphotransferase system (PTSGut). The gut operon is transcribed as a polycistronic gutFRMCBA messenger, the expression of which is induced by sorbitol and repressed by glucose. gutR encodes a transcriptional regulator with two PTS-regulated domains, a galactitol-specific EIIB-like domain (EIIBGat domain) and a mannitol/fructose-specific EIIA-like domain (EIIAMtl domain). Its inactivation abolished gut operon transcription and sorbitol uptake, indicating that it acts as a transcriptional activator. In contrast, cells carrying a gutB mutation expressed the gut operon constitutively, but they failed to transport sorbitol, indicating that EIIBCGut negatively regulates GutR. A footprint analysis showed that GutR binds to a 35-bp sequence upstream from the gut promoter. A sequence comparison with the presumed promoter region of gut operons from various firmicutes revealed a GutR consensus motif that includes an inverted repeat. The regulation mechanism of the L. casei gut operon is therefore likely to be operative in other firmicutes. Finally, gutM codes for a conserved protein of unknown function present in all sequenced gut operons. A gutM mutant, the first constructed in a firmicute, showed drastically reduced gut operon expression and sorbitol uptake, indicating a regulatory role also for GutM. PMID:18676710

  7. Regulation of Lactobacillus casei sorbitol utilization genes requires DNA-binding transcriptional activator GutR and the conserved protein GutM.

    PubMed

    Alcántara, Cristina; Sarmiento-Rubiano, Luz Adriana; Monedero, Vicente; Deutscher, Josef; Pérez-Martínez, Gaspar; Yebra, María J

    2008-09-01

    Sequence analysis of the five genes (gutRMCBA) downstream from the previously described sorbitol-6-phosphate dehydrogenase-encoding Lactobacillus casei gutF gene revealed that they constitute a sorbitol (glucitol) utilization operon. The gutRM genes encode putative regulators, while the gutCBA genes encode the EIIC, EIIBC, and EIIA proteins of a phosphoenolpyruvate-dependent sorbitol phosphotransferase system (PTS(Gut)). The gut operon is transcribed as a polycistronic gutFRMCBA messenger, the expression of which is induced by sorbitol and repressed by glucose. gutR encodes a transcriptional regulator with two PTS-regulated domains, a galactitol-specific EIIB-like domain (EIIB(Gat) domain) and a mannitol/fructose-specific EIIA-like domain (EIIA(Mtl) domain). Its inactivation abolished gut operon transcription and sorbitol uptake, indicating that it acts as a transcriptional activator. In contrast, cells carrying a gutB mutation expressed the gut operon constitutively, but they failed to transport sorbitol, indicating that EIIBC(Gut) negatively regulates GutR. A footprint analysis showed that GutR binds to a 35-bp sequence upstream from the gut promoter. A sequence comparison with the presumed promoter region of gut operons from various firmicutes revealed a GutR consensus motif that includes an inverted repeat. The regulation mechanism of the L. casei gut operon is therefore likely to be operative in other firmicutes. Finally, gutM codes for a conserved protein of unknown function present in all sequenced gut operons. A gutM mutant, the first constructed in a firmicute, showed drastically reduced gut operon expression and sorbitol uptake, indicating a regulatory role also for GutM.

  8. Identification and expression profiling analysis of calmodulin-binding transcription activator genes in maize (Zea mays L.) under abiotic and biotic stresses.

    PubMed

    Yue, Runqing; Lu, Caixia; Sun, Tao; Peng, Tingting; Han, Xiaohua; Qi, Jianshuang; Yan, Shufeng; Tie, Shuanggui

    2015-01-01

    The calmodulin-binding transcription activators (CAMTA) play critical roles in plant growth and responses to environmental stimuli. However, how CAMTAs function in responses to abiotic and biotic stresses in maize (Zea mays L.) is largely unknown. In this study, we first identified all the CAMTA homologous genes in the whole genome of maize. The results showed that nine ZmCAMTA genes showed highly diversified gene structures and tissue-specific expression patterns. Many ZmCAMTA genes displayed high expression levels in the roots. We then surveyed the distribution of stress-related cis-regulatory elements in the -1.5 kb promoter regions of ZmCAMTA genes. Notably, a large number of stress-related elements present in the promoter regions of some ZmCAMTA genes, indicating a genetic basis of stress expression regulation of these genes. Quantitative real-time PCR was used to test the expression of ZmCAMTA genes under several abiotic stresses (drought, salt, and cold), various stress-related hormones [abscisic acid, auxin, salicylic acid (SA), and jasmonic acid] and biotic stress [rice black-streaked dwarf virus (RBSDV) infection]. Furthermore, the expression pattern of ZmCAMTA genes under RBSDV infection was analyzed to investigate their potential roles in responses of different maize cultivated varieties to RBSDV. The expression of most ZmCAMTA genes responded to both abiotic and biotic stresses. The data will help us to understand the roles of CAMTA-mediated Ca(2+) signaling in maize tolerance to environmental stresses.

  9. Transcriptional regulation of the mouse alpha A-crystallin gene: activation dependent on a cyclic AMP-responsive element (DE1/CRE) and a Pax-6-binding site.

    PubMed Central

    Cvekl, A; Kashanchi, F; Sax, C M; Brady, J N; Piatigorsky, J

    1995-01-01

    Two cis-acting promoter elements (-108 to -100 and -49 to -33) of the mouse alpha A-crystallin gene, which is highly expressed in the ocular lens, were studied. Here we show that DE1 (-108 to -100; 5'TGACGGTG3'), which resembles the consensus cyclic AMP (cAMP)-responsive element sequence (CRE; 5'TGACGT[A/C][A/G]3'), behaves like a functional CRE site. Transfection experiments and electrophoretic mobility shift assays (EMSAs) using site-specific mutations correlated a loss of function with deviations from the CRE consensus sequence. Results of EMSAs in the presence of antisera against CREB, delta CREB, and CREM were consistent with the binding of CREB-like proteins to the DE1 sequence. Stimulation of alpha A-crystallin promoter activity via 8-bromo-cAMP, forskolin, or human T-cell leukemia virus type I Tax1 in transfections and reduction of activity of this site in cell-free transcription tests by competition with the somatostatin CRE supported the idea that DE1 is a functional CRE. Finally, Pax-6, a member of the paired-box family of transcription factors, activated the mouse alpha A-crystallin promoter in cotransfected COP-8 fibroblasts and bound to the -59 to -29 promoter sequence in EMSAs. These data provide evidence for a synergistic role of Pax-6 and CREB-like proteins for high expression of the mouse alpha A-crystallin gene in the lens. PMID:7823934

  10. Identification of the specific sequence recognized by Penicillium citrinum MlcR, a GAL4-type transcriptional activator of ML-236B (compactin) biosynthetic genes.

    PubMed

    Baba, S; Nihira, T; Hosobuchi, M

    2008-09-01

    MlcR is a pathway-specific transcriptional activator of the ML-236B biosynthetic genes in Penicillium citrinum. The MlcR-binding sequences were identified by an in vitro gel-shift assay and an in vivo reporter assay for the region between mlcA and mlcC as a model. The gel-shift assay showed that recombinant MlcR bound to the DNA sequence 5'-ACGGCGTTATTCGG-3' and most of the bases in this motif were required for the interaction between MlcR and DNA. In the reporter assay using beta-glucuronidase (GUS), substitution of the bases in this binding sequence resulted in the drastic reduction of GUS activities. These data clearly indicate that this MlcR-binding sequence is essential for the transcriptional activation of mlcA and mlcC in P. citrinum. Similar motifs were found in other loci of the ML-236B biosynthetic gene cluster and the consensus-binding motif for MlcR was predicted to be a direct repeat, 5'-WCGG-N(6)-TCGG-3'.

  11. Constitutive transcription of the human interleukin-6 gene by rheumatoid synoviocytes: spontaneous activation of NF-kappaB and CBF1.

    PubMed Central

    Miyazawa, K.; Mori, A.; Yamamoto, K.; Okudaira, H.

    1998-01-01

    The involvement of IL-6 in the pathogenesis of rheumatoid arthritis (RA) has been recently demonstrated. In the present study, we investigated the cellular and molecular mechanisms involved in the spontaneous IL-6 production by the fibroblast-like synoviocytes (FLSs) obtained from patients with RA. Cloned FLSs were established from the bulk cultures of FLSs by the limiting dilution method. Some FLS clones spontaneously produced large amounts of IL-6, whereas others produced low amounts of it. Neither anti-human TNF-alpha nor IL-1 antibody affected spontaneous IL-6 production of these FLS clones, suggesting that IL-6 production of the FLSs was endogenously up-regulated. A luciferase reporter plasmid containing the human IL-6 promoter region was significantly transcribed when transfected into the IL-6 high-producing clones, indicating that the rheumatoid FLSs retained constitutive transcriptional activity of the IL-6 gene. Electrophoretic mobility shift assays revealed that the binding activity of p50 and p65 NF-kappaB subunits and CBF1 was significantly enhanced in the IL-6 high-producing clones compared with that of IL-6 low-producing clones and cultured sarcoma cells, suggesting that spontaneous activation of NF-kappaB and CBF1 may lead to the constitutive transcription of the IL-6 gene by rheumatoid FLSs. Images Figure 2 Figure 5 Figure 6 Figure 7 PMID:9502421

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

    PubMed

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

    2017-03-16

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

  13. NonO binds to the CpG island of oct4 promoter and functions as a transcriptional activator of oct4 gene expression.

    PubMed

    Park, Yoojin; Lee, Ja-Myong; Hwang, Min-Young; Son, Gi-hoon; Geum, Dongho

    2013-01-01

    We investigated the relationship between oct4 gene expression patterns and CpG sites methylation profiles during ES cell differentiation into neurons, and identified relevant binding factor. The oct4 gene expression level gradually declined as ES cell differentiation progressed, and the CpG sites in the oct4 proximal enhancer (PE) and promoter regions were methylated in concert with ES cell differentiation. An electro-mobility shift assay (EMSA) showed that putative proteins bind to CpG sites in the oct4 PE/promoter. We purified CpG binding proteins with DNAbinding purification method, and NonO was identified by liquid chromatography-mass spectrometry. EMSA with specific competitors revealed that NonO specifically binds to the conserved CCGGTGAC sequence in the oct4 promoter. Methylation at a specific cytosine residue (CC* GGTGAC) reduced the binding affinity of NonO for the recognition sequence. Chromatin immunoprecipitation analysis confirmed that NonO binds to the unmethylated oct4 promoter. There were no changes in the NonO mRNA and protein levels between ES cells and differentiated cells. The transcriptional role of NonO in oct4 gene expression was evaluated by luciferase assays and knockdown experiments. The luciferase activity significantly increased threefold when the NonO expression vector was cotransfected with the NonO recognition sequence, indicating that NonO has a transcription activator effect on oct4 gene expression. In accordance with this effect, when NonO expression was inhibited by siRNA treatment, oct4 expression was also significantly reduced. In summary, we purified NonO, a novel protein that binds to the CpG island of oct4 promoter, and positively regulates oct4 gene expression in ES cells.

  14. Functional Analysis of 14 Genes That Constitute the Purine Catabolic Pathway in Bacillus subtilis and Evidence for a Novel Regulon Controlled by the PucR Transcription Activator

    PubMed Central

    Schultz, Anna C.; Nygaard, Per; Saxild, Hans H.

    2001-01-01

    The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the utilization of purines as nitrogen source in B. subtilis. Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires expression of five genes (pucA, pucB, pucC, pucD, and pucE). Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK. Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded by pucF. In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285° on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions). Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product. Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3- to 10-fold increase in expression was seen for most of the genes. However, expression of the pucABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B

  15. Topologies for perfect adaptation in gene transcription

    NASA Astrophysics Data System (ADS)

    Shi, Wenjia; Tang, Chao

    2014-03-01

    Adaptation is commonly used in sensory systems and signaling networks to allow the detection of further stimuli. Despite enzymatic network topologies for adaptation have been investigated systematically, the topology of transcriptional network that could perform adaptation still remains unclear, due to the complexity of transcriptional regulation. Here, we systematically investigated all three-node transcriptional networks, and found the topologies of transcriptional networks for adaptation are different from that of enzymatic ones. While both negative feedback loop (NFBL) and incoherent feed forward loop (IFFL) are capable of performing adaptation analytically, a positive self-regulation on buffer node is necessary for NFBL topology and more flexible structures emerge for IFFL than that of enzymatic networks. Most of the simulation results agree with analytical predictions. This study may explain the mechanism of adapted gene regulation behavior and supply a design table for gene regulatory adaptation.

  16. ZBRK1, a novel tumor suppressor, activates VHL gene transcription through formation of a complex with VHL and p300 in renal cancer.

    PubMed

    Chen, Ke; Yu, Gan; Gumireddy, Kiranmai; Li, Anping; Yao, Weimin; Gao, Lu; Chen, Shuliang; Hao, Jun; Wang, Ji; Huang, Qihong; Xu, Hua; Ye, Zhangqun

    2015-03-30

    Inactivation or mutation of the VHL gene causes various tumors, including clear cell renal cell carcinoma (ccRCC). In the present study, we identified ZBRK1 as a novel VHL interacting protein by yeast two-hybrid screening, and found a single ZBRK1-binding site located in the VHL promoter region. Ectopic expression of ZBRK1 increases transcriptional activity of the VHL, whereas the depletion of endogenous ZBRK1 by shRNA leads to reduction of VHL expression. We also demonstrate that the inhibition of VEGF transcription by ZBRK1 overexpression is dependent on VHL/HIF pathway. Moreover, VHL is confirmed to serve as a bridge component for the association of ZBRK1 and p300, which leads to an increase in ZBRK1 transcriptional activity in the VHL promoter. We further provide striking evidences that ZBRK1 acts as a tumor suppressor in renal carcinoma by a variety of in vitro and in vivo assays, and ZBRK1 may represent a molecular marker to distinguish patients with ccRCC at high risk from those with a better survival prognosis. Taken together, these findings suggest that ZBRK1 suppresses renal cancer progression perhaps by regulating VHL expression.

  17. ZBRK1, a novel tumor suppressor, activates VHL gene transcription through formation of a complex with VHL and p300 in renal cancer

    PubMed Central

    Gumireddy, Kiranmai; Li, Anping; Yao, Weimin; Gao, Lu; Chen, Shuliang; Hao, Jun; Wang, Ji; Huang, Qihong; Xu, Hua; Ye, Zhangqun

    2015-01-01

    Inactivation or mutation of the VHL gene causes various tumors, including clear cell renal cell carcinoma (ccRCC). In the present study, we identified ZBRK1 as a novel VHL interacting protein by yeast two-hybrid screening, and found a single ZBRK1-binding site located in the VHL promoter region. Ectopic expression of ZBRK1 increases transcriptional activity of the VHL, whereas the depletion of endogenous ZBRK1 by shRNA leads to reduction of VHL expression. We also demonstrate that the inhibition of VEGF transcription by ZBRK1 overexpression is dependent on VHL/HIF pathway. Moreover, VHL is confirmed to serve as a bridge component for the association of ZBRK1 and p300, which leads to an increase in ZBRK1 transcriptional activity in the VHL promoter. We further provide striking evidences that ZBRK1 acts as a tumor suppressor in renal carcinoma by a variety of in vitro and in vivo assays, and ZBRK1 may represent a molecular marker to distinguish patients with ccRCC at high risk from those with a better survival prognosis. Taken together, these findings suggest that ZBRK1 suppresses renal cancer progression perhaps by regulating VHL expression. PMID:25749518

  18. Gene transcription and electromagnetic fields

    SciTech Connect

    Henderson, A.S.

    1992-01-01

    Our overall aim is to obtain sufficient information to allow us to ultimately determine whether ELF EM field exposure is an initiating factor in neoplastic transformation and/or if exposure can mimic characteristics of the second-step counterpart in neoplastic disease. This aim is based on our previous findings that levels of some transcripts are increased in cells exposed to EM fields. While the research is basic in nature, the ramifications have bearing on the general safety of exposure to EM fields in industrial and everyday life. A large array of diverse biological effects are reported to occur as the result of exposure to elf EM fields, suggesting that the cell response to EM fields is at a basic level, presumably initiated by molecular and/or biophysical events at the cell membrane. The hypothesized route is a signal transduction pathway involving membrane calcium fluxes. Information flow resulting from signal transduction can mediate the induction of regulatory factors in the cell, and directly affect how transcription is regulated.

  19. Transcriptional responses to loss or gain of function of the leucine-rich repeat kinase 2 (LRRK2) gene uncover biological processes modulated by LRRK2 activity

    PubMed Central

    Nikonova, Elena V.; Xiong, Yulan; Tanis, Keith Q.; Dawson, Valina L.; Vogel, Robert L.; Finney, Eva M.; Stone, David J.; Reynolds, Ian J.; Kern, Jonathan T.; Dawson, Ted M.

    2012-01-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common genetic cause of Parkinson's disease (PD) and cause both autosomal dominant familial and sporadic PD. Currently, the physiological and pathogenic activities of LRRK2 are poorly understood. To decipher the biological functions of LRRK2, including the genes and pathways modulated by LRRK2 kinase activity in vivo, we assayed genome-wide mRNA expression in the brain and peripheral tissues from LRRK2 knockout (KO) and kinase hyperactive G2019S (G2019S) transgenic mice. Subtle but significant differences in mRNA expression were observed relative to wild-type (WT) controls in the cortex, striatum and kidney of KO animals, but only in the striatum in the G2019S model. In contrast, robust, consistent and highly significant differences were identified by the direct comparison of KO and G2019S profiles in the cortex, striatum, kidney and muscle, indicating opposite effects on mRNA expression by the two models relative to WT. Ribosomal and glycolytic biological functions were consistently and significantly up-regulated in LRRK2 G2019S compared with LRRK2 KO tissues. Genes involved in membrane-bound organelles, oxidative phosphorylation, mRNA processing and the endoplasmic reticulum were down-regulated in LRRK2 G2019S mice compared with KO. We confirmed the expression patterns of 35 LRRK2-regulated genes using quantitative reverse transcription polymerase chain reaction. These findings provide the first description of the transcriptional responses to genetically modified LRRK2 activity and provide preclinical target engagement and/or pharmacodynamic biomarker strategies for LRRK2 and may inform future therapeutic strategies for LRRK2-associated PD. PMID:21972245

  20. Aeromonas hydrophila Lateral Flagellar Gene Transcriptional Hierarchy

    PubMed Central

    Wilhelms, Markus; Gonzalez, Victor; Merino, Susana

    2013-01-01

    Aeromonas hydrophila AH-3 lateral flagella are not assembled when bacteria grow in liquid media; however, lateral flagellar genes are transcribed. Our results indicate that A. hydrophila lateral flagellar genes are transcribed at three levels (class I to III genes) and share some similarities with, but have many important differences from, genes of Vibrio parahaemolyticus. A. hydrophila lateral flagellum class I gene transcription is σ70 dependent, which is consistent with the fact that lateral flagellum is constitutively transcribed, in contrast to the characteristics of V. parahaemolyticus. The fact that multiple genes are included in class I highlights that lateral flagellar genes are less hierarchically transcribed than polar flagellum genes. The A. hydrophila lafK-fliEJL gene cluster (where the subscript L distinguishes genes for lateral flagella from those for polar flagella) is exclusively from class I and is in V. parahaemolyticus class I and II. Furthermore, the A. hydrophila flgAMNL cluster is not transcribed from the σ54/LafK-dependent promoter and does not contain class II genes. Here, we propose a gene transcriptional hierarchy for the A. hydrophila lateral flagella. PMID:23335410

  1. The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors.

    PubMed Central

    Antinore, M J; Birrer, M J; Patel, D; Nader, L; McCance, D J

    1996-01-01

    The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16-induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos. The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc-finger motif, but not the pRb binding domain, is involved in these interactions. Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun. E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway. Images PMID:8617242

  2. Human papillomavirus 16 oncoprotein E7 stimulates UBF1-mediated rDNA gene transcription, inhibiting a p53-independent activity of p14ARF.

    PubMed

    Dichamp, Isabelle; Séité, Paule; Agius, Gérard; Barbarin, Alice; Beby-Defaux, Agnès

    2014-01-01

    High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14ARF-p53 pathway. pRb and p14ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14ARF, UBF1 and E7, although p14ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19ARF, the mouse homologue of p14ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14ARF. These results point to a mutually functional interaction between p14ARF and E7 that might partly explain why the sustained p14ARF expression observed in most cervical pre-malignant lesions and

  3. Gene looping facilitates TFIIH kinase-mediated termination of transcription

    PubMed Central

    Medler, Scott; Ansari, Athar

    2015-01-01

    TFIIH is a general transcription factor with kinase and helicase activities. The kinase activity resides in the Kin28 subunit of TFIIH. The role of Kin28 kinase in the early steps of transcription is well established. Here we report a novel role of Kin28 in the termination of transcription. We show that RNAPII reads through a termination signal upon kinase inhibition. Furthermore, the recruitment of termination factors towards the 3′ end of a gene was compromised in the kinase mutant, thus confirming the termination defect. A concomitant decrease in crosslinking of termination factors near the 5′ end of genes was also observed in the kinase-defective mutant. Simultaneous presence of termination factors towards both the ends of a gene is indicative of gene looping; while the loss of termination factor occupancy from the distal ends suggest the abolition of a looped gene conformation. Accordingly, CCC analysis revealed that the looped architecture of genes was severely compromised in the Kin28 kinase mutant. In a looping defective sua7-1 mutant, even the enzymatically active Kin28 kinase could not rescue the termination defect. These results strongly suggest a crucial role of Kin28 kinase-dependent gene looping in the termination of transcription in budding yeast. PMID:26286112

  4. Regulation of cytokine gene transcription in the immune system.

    PubMed

    Holloway, A F; Rao, S; Shannon, M F

    2002-01-01

    The controlled expression of cytokine genes is an essential component of an immune response. The specific types of cytokines as well as the time and place of their production is important in generating an appropriate immune response to an infectious agent. Aberrant expression is associated with pathological conditions of the immune system such as autoimmunity, atopy and chronic inflammation. Cytokine gene transcription is generally induced in a cell-specific manner. Over the last 15 years, a large amount of information has been generated describing the transcriptional controls that are exerted on cytokine genes. Recently, efforts have been directed at understanding how these genes are transcribed in a chromatin context. This review will discuss the mechanisms by which cytokine genes become available for transcription in a cell-restricted manner as well as the mechanisms by which these genes sense their environment and activate high level transcription in a transient manner. Particular attention will be paid to the role of chromatin in allowing transcription factor access to appropriate genes.

  5. Novel [NiFe]- and [FeFe]-hydrogenase gene transcripts indicative of active facultative aerobes and obligate anaerobes in earthworm gut contents.

    PubMed

    Schmidt, Oliver; Wüst, Pia K; Hellmuth, Susanne; Borst, Katharina; Horn, Marcus A; Drake, Harold L

    2011-09-01

    The concomitant occurrence of molecular hydrogen (H(2)) and organic acids along the alimentary canal of the earthworm is indicative of ongoing fermentation during gut passage. Fermentative H(2) production is catalyzed by [FeFe]-hydrogenases and group 4 [NiFe]-hydrogenases in obligate anaerobes (e.g., Clostridiales) and facultative aerobes (e.g., Enterobacteriaceae), respectively, functional groups that might respond differently to contrasting redox conditions. Thus, the objectives of this study were to assess the redox potentials of the alimentary canal of Lumbricus terrestris and analyze the hydrogenase transcript diversities of H(2) producers in glucose-supplemented gut content microcosms. Although redox potentials in the core of the alimentary canal were variable on an individual worm basis, average redox potentials were similar. The lowest redox potentials occurred in the foregut and midgut regions, averaging 40 and 110 mV, respectively. Correlation plots between hydrogenase amino acid sequences and 16S rRNA gene sequences indicated that closely related hydrogenases belonged to closely related taxa, whereas distantly related hydrogenases did not necessarily belong to distantly related taxa. Of 178 [FeFe]-hydrogenase gene transcripts, 177 clustered in 12 Clostridiales-affiliated operational taxonomic units, the majority of which were indicative of heretofore unknown hydrogenases. Of 86 group 4 [NiFe]-hydrogenase gene transcripts, 79% and 21% were affiliated with organisms in the Enterobacteriaceae and Aeromonadaceae, respectively. The collective results (i) suggest that fermenters must cope with variable and moderately oxidative redox conditions along the alimentary canal, (ii) demonstrate that heretofore undetected hydrogenases are present in the earthworm gut, and (iii) corroborate previous findings implicating Clostridiaceae and Enterobacteriaceae as active fermentative taxa in earthworm gut content.

  6. Enhanced DNA repair of bleomycin-induced 3'-phosphoglycolate termini at the transcription start sites of actively transcribed genes in human cells.

    PubMed

    Murray, Vincent; Chen, Jon K; Galea, Anne M

    2014-11-01

    The anti-tumour agent, bleomycin, cleaves DNA to give 3'-phosphoglycolate and 5'-phosphate termini. The removal of 3'-phosphoglycolate to give 3'-OH ends is a very important step in the DNA repair of these lesions. In this study, next-generation DNA sequencing was utilised to investigate the repair of these 3'-phosphoglycolate termini at the transcription start sites (TSSs) of genes in HeLa cells. The 143,600 identified human TSSs in HeLa cells comprised 82,596 non-transcribed genes and 61,004 transcribed genes; and the transcribed genes were divided into quintiles of 12,201 genes comprising the top 20%, 20-40%, 40-60%, 60-80%, 80-100% of expressed genes. Repair of bleomycin-induced 3'-phosphoglycolate termini was enhanced at actively transcribed genes. The top 20% and 20-40% quintiles had a very similar level of enhanced repair, the 40-60% quintile was intermediate, while the 60-80% and 80-100% quintiles were close to the low level of enhancement found in non-transcribed genes. There were also interesting differences regarding bleomycin repair on the sense and antisense strands of DNA at TSSs. The sense strand had highly enhanced repair between 0 and 250bp relative to the TSS, while for the antisense strand highly enhanced repair was between 150 and 450bp. Repair of DNA damage is a major mechanism of resistance to anti-tumour drugs and this study provides an insight into this process in human tumour cells.

  7. Cooperative activation of Xenopus rhodopsin transcription by paired-like transcription factors

    PubMed Central

    2014-01-01

    Background In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking. Results We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element. Conclusions We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription. PMID:24499263

  8. Entinostat up-regulates the CAMP gene encoding LL-37 via activation of STAT3 and HIF-1α transcription factors

    PubMed Central

    Miraglia, Erica; Nylén, Frank; Johansson, Katarina; Arnér, Elias; Cebula, Marcus; Farmand, Susan; Ottosson, Håkan; Strömberg, Roger; Gudmundsson, Gudmundur H.; Agerberth, Birgitta; Bergman, Peter

    2016-01-01

    Bacterial resistance against classical antibiotics is a growing problem and the development of new antibiotics is limited. Thus, novel alternatives to antibiotics are warranted. Antimicrobial peptides (AMPs) are effector molecules of innate immunity that can be induced by several compounds, including vitamin D and phenyl-butyrate (PBA). Utilizing a luciferase based assay, we recently discovered that the histone deacetylase inhibitor Entinostat is a potent inducer of the CAMP gene encoding the human cathelicidin LL-37. Here we investigate a mechanism for the induction and also find that Entinostat up-regulates human β-defensin 1. Analysis of the CAMP promoter sequence revealed binding sites for the transcription factors STAT3 and HIF-1α. By using short hairpin RNA and selective inhibitors, we found that both transcription factors are involved in Entinostat-induced expression of LL-37. However, only HIF-1α was found to be recruited to the CAMP promoter, suggesting that Entinostat activates STAT3, which promotes transcription of CAMP by increasing the expression of HIF-1α. Finally, we provide in vivo relevance to our findings by showing that Entinostat-elicited LL-37 expression was impaired in macrophages from a patient with a STAT3-mutation. Combined, our findings support a role for STAT3 and HIF-1α in the regulation of LL-37 expression. PMID:27633343

  9. Vgl-4, a novel member of the vestigial-like family of transcription cofactors, regulates alpha1-adrenergic activation of gene expression in cardiac myocytes.

    PubMed

    Chen, Hsiao-Huei; Mullett, Steven J; Stewart, Alexandre F R

    2004-07-16

    Cardiac and skeletal muscle genes are regulated by the transcriptional enhancer factor (TEF-1) family of transcription factors. In skeletal muscle, TEF-1 factors interact with a skeletal muscle-specific cofactor called Vestigial-like 2 (Vgl-2) that is related to the Drosophila protein Vestigial. Here, we characterize Vgl-4, the only member of the Vestigial-like family expressed in the heart. Unlike other members of the Vgl family that have a single TEF-1 interaction domain called the tondu (TDU) motif, Vgl-4 has two TDU motifs in its carboxyl-terminal domain. Like other Vgl factors, Vgl-4 physically interacts with TEF-1 in an immunoprecipitation assay. Vgl-4 functionally interacts with TEF-1 and also with myocyte enhancer factor 2 in a mammalian two-hybrid assay. Overexpression of Vgl-4 in cardiac myocytes interfered with the basal expression and alpha1-adrenergic receptor-dependent activation of a TEF-1-dependent skeletal alpha-actin promoter. In cardiac myocytes cultured in serum and in serum-free medium, a myc-tagged Vgl-4 protein was located in the nucleus and cytoplasm but was exported from the nucleus when cells were treated with alpha1-adrenergic receptor agonist. A chimeric nuclear-retained Vgl-4 protein inhibited alpha1-adrenergic receptor-dependent activation. In contrast, deletion of the TDU motifs of Vgl-4 prevented Vgl-4 nuclear localization, relieved Vgl-4 interference of basal activity, and enhanced alpha1-adrenergic up-regulation of the skeletal alpha-actin promoter. Nuclear export of Vgl-4 is dependent on the nuclear exportin CRM-1. These results suggest that Vgl-4 modulates the activity of TEF-1 factors and counteracts alpha1-adrenergic activation of gene expression in cardiac myocytes.

  10. Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

    PubMed

    Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

    2009-02-01

    Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

  11. API2-MALT1 fusion protein induces transcriptional activation of the API2 gene through NF-{kappa}B binding elements: Evidence for a positive feed-back loop pathway resulting in unremitting NF-{kappa}B activation

    SciTech Connect

    Hosokawa, Yoshitaka . E-mail: yhosokaw@aichi-cc.jp; Suzuki, Hiroko; Nakagawa, Masao; Lee, Tae H.; Seto, Masao

    2005-08-19

    t(11;18)(q21;q21) is a characteristic as well as the most frequent chromosomal translocation in mucosa-associated lymphoid tissue (MALT) type lymphoma, and this translocation results in a fusion transcript, API2-MALT1. Although API2-MALT1 has been shown to enforce activation of NF-{kappa}B signaling, the transcriptional target genes of this fusion protein remains to be identified. Our analyses of the API2-MALT transfectants suggested that one of the target genes may be the apoptotic inhibitor API2 gene. Luciferase reporter assays with deletion and mutational constructs of the API2 promoter and electrophoretic mobility shift assays established that API2-MALT1 induces transcriptional activation of the API2 gene through two NF-{kappa}B binding elements. Moreover, supershift experiments indicated that these elements are recognized by the NF-{kappa}B p50/p65 heterodimer. Taken together, our results strongly indicated that API2-MALT1 possesses a novel mechanism of self-activation by up-regulating its own expression in t(11;18)(q21;q21)-carrying MALT lymphomas, highlighting a positive feedback-loop pathway resulting in unremitting NF-{kappa}B activation.

  12. The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATP-dependent ubiquitin-proteasome pathway.

    PubMed Central

    Bailey, J L; Wang, X; England, B K; Price, S R; Ding, X; Mitch, W E

    1996-01-01

    Chronic renal failure (CRF) is associated with negative nitrogen balance and loss of lean body mass. To identify specific proteolytic pathways activated by CRF, protein degradation was measured in incubated epitrochlearis muscles from CRF and sham-operated, pair-fed rats. CRF stimulated muscle proteolysis, and inhibition of lysosomal and calcium-activated proteases did not eliminate this increase. When ATP production was blocked, proteolysis in CRF muscles fell to the same level as that in control muscles. Increased proteolysis was also prevented by feeding CRF rats sodium bicarbonate, suggesting that activation depends on acidification. Evidence that the ATP-dependent ubiquitin-proteasome pathway is stimulated by the acidemia of CRF includes the following findings: (a) An inhibitor of the proteasome eliminated the increase in muscle proteolysis; and (b) there was an increase in mRNAs encoding ubiquitin (324%) and proteasome subunits C3 (137%) and C9 (251%) in muscle. This response involved gene activation since transcription of mRNAs for ubiquitin and the C3 subunit were selectively increased in muscle of CRF rats. We conclude that CRF stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent pathway. The mechanism depends on acidification and increased expression of genes encoding components of the system. These responses could contribute to the loss of muscle mass associated with CRF. PMID:8617877

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

    PubMed Central

    Prywes, R; Zhu, H

    1992-01-01

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

  14. Heterodimeric Drosophila gap gene protein complexes acting as transcriptional repressors.

    PubMed Central

    Sauer, F; Jäckle, H

    1995-01-01

    The Drosophila gap gene Krüppel (Kr) encodes a transcriptional regulator. It acts both as an integral part of the Drosophila segmentation gene in the early blastoderm and in a variety of tissues and organs at later stages of embryogenesis. In transfected tissue culture cells, the Kr protein (Kr) was shown to both activate and repress gene expression in a concentration-dependent manner when acting from a single binding site close to the promoter. Here we show that KR can associate with the transcription factors encoded by the gap genes knirps (kni) and hunchback (hb) which affect KR-dependent gene expression in Drosophila tissue culture cells. The association of DNA-bound hb protein or free kni protein with distinct but different regions of KR results in the formation of DNA-bound transcriptional repressor complexes. Our results suggest that individual transcription factors can associate to form protein complexes which act as direct repressors of transcription. The interactions shown here add an unexpected level of complexity to the control of gene expression. Images PMID:7588607

  15. Constitutive androstane receptor transcriptionally activates human CYP1A1 and CYP1A2 genes through a common regulatory element in the 5'-flanking region.

    PubMed

    Yoshinari, Kouichi; Yoda, Noriaki; Toriyabe, Takayoshi; Yamazoe, Yasushi

    2010-01-15

    Phenobarbital has long been known to increase cellular levels of CYP1A1 and CYP1A2 possibly through a pathway(s) independent of aryl hydrocarbon receptor. We have investigated the role of constitutive androstane receptor (CAR), a xenobiotic-responsive nuclear receptor, in the transactivation of human CYP1A1 and CYP1A2. These genes are located in a head-to-head orientation, sharing a 5'-flanking region. Reporter assays were thus performed with dual-reporter constructs, containing the whole or partially deleted human CYP1A promoter between two different reporter genes. In this system, human CAR (hCAR) enhanced the transcription of both genes through common promoter regions from -461 to -554 and from -18089 to -21975 of CYP1A1. With reporter assays using additional deleted and mutated constructs, electrophoresis mobility shift assays and chromatin immunoprecipitation assays, an ER8 motif (everted repeat separated by eight nucleotides), located at around -520 of CYP1A1, was identified as an hCAR-responsive element and a binding motif of hCAR/human retinoid X receptor alpha heterodimer. hCAR enhanced the transcription of both genes also in the presence of an aryl hydrocarbon receptor ligand. Finally, hCAR activation increased CYP1A1 and CYP1A2 mRNA levels in cultured human hepatocytes. Our results indicate that CAR transactivates human CYP1A1 and CYP1A2 in human hepatocytes through the common cis-element ER8. Interestingly, the ER8 motif is highly conserved in the CYP1A1 proximal promoter sequences of various species, suggesting a fundamental role of CAR in the xenobiotic-induced expression of CYP1A1 and CYP1A2 independent of aryl hydrocarbon receptor.

  16. Transcriptional enhancer from milk protein genes

    DOEpatents

    Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

    1999-01-01

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  17. Transcriptional enhancer from milk protein genes

    SciTech Connect

    Casperson, G.F.; Schmidhauser, C.T.; Bissell, M.J.

    1999-12-21

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  18. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    SciTech Connect

    Tennyson, C.N.; Worton, R.G.

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

  19. Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor.

    PubMed

    Paonessa, Francesco; Criscuolo, Stefania; Sacchetti, Silvio; Amoroso, Davide; Scarongella, Helena; Pecoraro Bisogni, Federico; Carminati, Emanuele; Pruzzo, Giacomo; Maragliano, Luca; Cesca, Fabrizia; Benfenati, Fabio

    2016-01-05

    Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light-oxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na(+)-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na(+) currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases.

  20. Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

    PubMed Central

    Belacortu, Yaiza; Weiss, Ron; Kadener, Sebastian; Paricio, Nuria

    2012-01-01

    Background Cabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. Methodology/Principal Findings To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. Conclusions/Significance Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of

  1. Sequential Logic Model Deciphers Dynamic Transcriptional Control of Gene Expressions

    PubMed Central

    Yeo, Zhen Xuan; Wong, Sum Thai; Arjunan, Satya Nanda Vel; Piras, Vincent; Tomita, Masaru; Selvarajoo, Kumar; Giuliani, Alessandro; Tsuchiya, Masa

    2007-01-01

    Background Cellular signaling involves a sequence of events from ligand binding to membrane receptors through transcription factors activation and the induction of mRNA expression. The transcriptional-regulatory system plays a pivotal role in the control of gene expression. A novel computational approach to the study of gene regulation circuits is presented here. Methodology Based on the concept of finite state machine, which provides a discrete view of gene regulation, a novel sequential logic model (SLM) is developed to decipher control mechanisms of dynamic transcriptional regulation of gene expressions. The SLM technique is also used to systematically analyze the dynamic function of transcriptional inputs, the dependency and cooperativity, such as synergy effect, among the binding sites with respect to when, how much and how fast the gene of interest is expressed. Principal Findings SLM is verified by a set of well studied expression data on endo16 of Strongylocentrotus purpuratus (sea urchin) during the embryonic midgut development. A dynamic regulatory mechanism for endo16 expression controlled by three binding sites, UI, R and Otx is identified and demonstrated to be consistent with experimental findings. Furthermore, we show that during transition from specification to differentiation in wild type endo16 expression profile, SLM reveals three binary activities are not sufficient to explain the transcriptional regulation of endo16 expression and additional activities of binding sites are required. Further analyses suggest detailed mechanism of R switch activity where indirect dependency occurs in between UI activity and R switch during specification to differentiation stage. Conclusions/Significance The sequential logic formalism allows for a simplification of regulation network dynamics going from a continuous to a discrete representation of gene activation in time. In effect our SLM is non-parametric and model-independent, yet providing rich biological

  2. The promoter of the white spot syndrome virus immediate-early gene WSSV108 is activated by the cellular KLF transcription factor.

    PubMed

    Liu, Wang-Jing; Lo, Chu-Fang; Kou, Guang-Hsiung; Leu, Jiann-Horng; Lai, Ying-Jang; Chang, Li-Kwan; Chang, Yun-Shiang

    2015-03-01

    A series of deletion and mutation assays of the white spot syndrome virus (WSSV) immediate-early gene WSSV108 promoter showed that a Krüppel-like factor (KLF) binding site located from -504 to -495 (relative to the transcription start site) is important for the overall level of WSSV108 promoter activity. Electrophoretic mobility shift assays further showed that overexpressed recombinant Penaeus monodon KLF (rPmKLF) formed a specific protein-DNA complex with the (32)P-labeled KLF binding site of the WSSV108 promoter, and that higher levels of Litopenaeus vannamei KLF (LvKLF) were expressed in WSSV-infected shrimp. A transactivation assay indicated that the WSSV108 promoter was strongly activated by rPmKLF in a dose-dependent manner. Lastly, we found that specific silencing of LvKLF expression in vivo by dsRNA injection dramatically reduced both WSSV108 expression and WSSV replication. We conclude that shrimp KLF is important for WSSV genome replication and gene expression, and that it binds to the WSSV108 promoter to enhance the expression of this immediate-early gene.

  3. Single-cell RNA sequencing highlights transcription activity of autophagy-related genes during hematopoietic stem cell formation in mouse embryos.

    PubMed

    Hu, Yongfei; Huang, Yan; Yi, Ying; Wang, Hongwei; Liu, Bing; Yu, Jia; Wang, Dong

    2017-04-03

    Accumulating evidence has demonstrated that macroautophagy/autophagy plays an essential role in self-renewal and differentiation in embryonic hematopoiesis. Here, according to the RNA sequencing data sets of 5 population cells related to hematopoietic stem cell (HSC) formation during mouse embryogenesis (endothelial cells, PTPRC/CD45(-) and PTPRC/CD45(+) pre-HSCs in the E11 aorta-gonad-mesonephros (AGM) region, mature HSCs in E12 and E14 fetal liver), we explored the dynamic expression of mouse autophagy-related genes in this course at the single-cell level. Our results revealed that the transcription activity of autophagy-related genes had a substantial increase when endothelial cells (ECs) specified into pre-HSCs, and the upregulation of autophagy-essential genes correlated with reduced NOTCH signaling in pre-HSCs, suggesting the autophagy activity may be greatly enhanced during pre-HSC specification from endothelial precursors. In summary, our results presented strong evidence that autophagy plays a critical role in HSC emergence during mouse midgestation.

  4. Transcriptional gene silencing as a tool for uncovering gene function in maize.

    PubMed

    Cigan, A Mark; Unger-Wallace, Erica; Haug-Collet, Kristin

    2005-09-01

    Transcriptional gene silencing has broad applications for studying gene function in planta. In maize, a large number of genes have been identified as tassel-preferred in their expression pattern, both by traditional genetic methods and by recent high-throughput expression profiling platforms. Approaches using RNA suppression may provide a rapid alternative means to identify genes directly related to pollen development in maize. The male fertility gene Ms45 and several anther-expressed genes of unknown function were used to evaluate the efficacy of generating male-sterile plants by transcriptional gene silencing. A high frequency of male-sterile plants was obtained by constitutively expressing inverted repeats (IR) of the Ms45 promoter. These sterile plants lacked MS45 mRNA due to transcriptional inactivity of the target promoter. Moreover, fertility was restored to these promoter IR-containing plants by expressing the Ms45 coding region using heterologous promoters. Transcriptional silencing of other anther-expressed genes also significantly affected male fertility phenotypes and led to increased methylation of the target promoter DNA sequences. These studies provide evidence of disruption of gene activity in monocots by RNA interference constructs directed against either native or transformed promoter regions. This approach not only enables the correlation of monocot anther-expressed genes with functions that are important for reproduction in maize, but may also provide a tool for studying gene function and identifying regulatory components unique to transcriptional gene control.

  5. Transcriptional Targeting in Cancer Gene Therapy

    PubMed Central

    2003-01-01

    Cancer gene therapy has been one of the most exciting areas of therapeutic research in the past decade. In this review, we discuss strategies to restrict transcription of transgenes to tumour cells. A range of promoters which are tissue-specific, tumour-specific, or inducible by exogenous agents are presented. Transcriptional targeting should prevent normal tissue toxicities associated with other cancer treatments, such as radiation and chemotherapy. In addition, the specificity of these strategies should provide improved targeting of metastatic tumours following systemic gene delivery. Rapid progress in the ability to specifically control transgenes will allow systemic gene delivery for cancer therapy to become a real possibility in the near future. PMID:12721516

  6. LIN-3/EGF promotes the programmed cell death of specific cells in Caenorhabditis elegans by transcriptional activation of the pro-apoptotic gene egl-1.

    PubMed

    Jiang, Hang-Shiang; Wu, Yi-Chun

    2014-08-01

    Programmed cell death (PCD) is the physiological death of a cell mediated by an intracellular suicide program. Although key components of the PCD execution pathway have been identified, how PCD is regulated during development is poorly understood. Here, we report that the epidermal growth factor (EGF)-like ligand LIN-3 acts as an extrinsic signal to promote the death of specific cells in Caenorhabditis elegans. The loss of LIN-3 or its receptor, LET-23, reduced the death of these cells, while excess LIN-3 or LET-23 signaling resulted in an increase in cell deaths. Our molecular and genetic data support the model that the LIN-3 signal is transduced through LET-23 to activate the LET-60/RAS-MPK-1/ERK MAPK pathway and the downstream ETS domain-containing transcription factor LIN-1. LIN-1 binds to, and activates transcription of, the key pro-apoptotic gene egl-1, which leads to the death of specific cells. Our results provide the first evidence that EGF induces PCD at the whole organism level and reveal the molecular basis for the death-promoting function of LIN-3/EGF. In addition, the level of LIN-3/EGF signaling is important for the precise fine-tuning of the life-versus-death fate. Our data and the previous cell culture studies that say EGF triggers apoptosis in some cell lines suggest that the EGF-mediated modulation of PCD is likely conserved in C. elegans and humans.

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

    PubMed

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

    2013-02-15

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. Transcriptional Activity of Gene Encoding Subunits R1 and R2 of Interferon Gamma Receptor in Peripheral Blood Mononuclear Cells in Patients with Slow Coronary Flow

    PubMed Central

    Faramarz-Gaznagh, Sanaz; Khadem-Ansari, Mohammad-Hasan; Seyed-Mohammadzad, Mir-Hossein; Bagheri, Morteza; Nemati, Mohadeseh; Shirpoor, Alireza; Saboori, Ehsan

    2016-01-01

    Summary Background Slow coronary flow (SCF) is a coronary artery disorder characterized with delayed opacification of epicardial coronary arteries without obstructive coronary disease. The pathophysiological mechanisms of SCF remain unclear. One of the possible mechanisms that may participate in the pathology of SCF is endothelial dysfunction related to the inflammatory process. Interferon gamma (IFN-γ) is an inflammatory cytokine that acts through its specific receptor composed of two subunits, IFN-γR1 and IFN-γR2. Transcriptional activity of the gene encoding these subunits influences IFN-γ activity. This study aimed to investigate the gene expression of IFN-γ receptor subunits in peripheral blood mononuclear cells (PBMC) from patients with SCF. Methods The study was performed with 30 patients (22 male/8 female) aged 35–76 (52.8±11.7 years) with SCF and 15 sex- (11 male/4 female), Body Max Index (BMI)- and age-matched (54.73±9.42 years) healthy subjects. Total mRNA was extracted from PBMC and was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The relative expression values (2-ΔΔCt) between control and case groups were determined and the Mann-Whitney U test was used for statistical analysis. Results There was a significant increase in the gene expression of IFN-γR1 in PBMC from SCF patients vs. controls (P< 0.0001); but the differences in IFN-γR2 gene expression were statistically insignificant between patient and control groups (P= 0.853). Conclusions It can be concluded that IFN-γ gene expression may influence the function of microvasculature and thereby contribute to the pathophysiology of SCF.

  10. Impact of ACTH Signaling on Transcriptional Regulation of Steroidogenic Genes

    PubMed Central

    Ruggiero, Carmen; Lalli, Enzo

    2016-01-01

    The trophic peptide hormone adrenocorticotropic (ACTH) stimulates steroid hormone biosynthesis evoking both a rapid, acute response and a long-term, chronic response, via the activation of cAMP/protein kinase A (PKA) signaling. The acute response is initiated by the mobilization of cholesterol from lipid stores and its delivery to the inner mitochondrial membrane, a process that is mediated by the steroidogenic acute regulatory protein. The chronic response results in the increased coordinated transcription of genes encoding steroidogenic enzymes. ACTH binding to its cognate receptor, melanocortin 2 receptor (MC2R), stimulates adenylyl cyclase, thus inducing cAMP production, PKA activation, and phosphorylation of specific nuclear factors, which bind to target promoters and facilitate coactivator protein recruitment to direct steroidogenic gene transcription. This review provides a general view of the transcriptional control exerted by the ACTH/cAMP system on the expression of genes encoding for steroidogenic enzymes in the adrenal cortex. Special emphasis will be given to the transcription factors required to mediate ACTH-dependent transcription of steroidogenic genes. PMID:27065945

  11. Insect Neuropeptide Bursicon Homodimers Induce Innate Immune and Stress Genes during Molting by Activating the NF-κB Transcription Factor Relish

    PubMed Central

    Li, Sheng; Gilbert, Lawrence I.; Stanley, David; Song, Qisheng

    2012-01-01

    Background Bursicon is a heterodimer neuropeptide composed of two cystine knot proteins, bursicon α (burs α) and bursicon β (burs β), that elicits cuticle tanning (melanization and sclerotization) through the Drosophila leucine-rich repeats-containing G protein-coupled receptor 2 (DLGR2). Recent studies show that both bursicon subunits also form homodimers. However, biological functions of the homodimers have remained unknown until now. Methodology/Principal Findings In this report, we show in Drosophila melanogaster that both bursicon homodimers induced expression of genes encoding antimicrobial peptides (AMPs) in neck-ligated adults following recombinant homodimer injection and in larvae fat body after incubation with recombinant homodimers. These AMP genes were also up-regulated in 24 h old unligated flies (when the endogenous bursicon level is low) after injection of recombinant homodimers. Up-regulation of AMP genes by the homodimers was accompanied by reduced bacterial populations in fly assay preparations. The induction of AMP expression is via activation of the NF-κB transcription factor Relish in the immune deficiency (Imd) pathway. The influence of bursicon homodimers on immune function does not appear to act through the heterodimer receptor DLGR2, i.e. novel receptors exist for the homodimers. Conclusions/Significance Our results reveal a mechanism of CNS-regulated prophylactic innate immunity during molting via induced expression of genes encoding AMPs and genes of the Turandot family. Turandot genes are also up-regulated by a broader range of extreme insults. From these data we infer that CNS-generated bursicon homodimers mediate innate prophylactic immunity to both stress and infection during the vulnerable molting cycle. PMID:22470576

  12. Inter-MAR association contributes to transcriptionally active looping events in human beta-globin gene cluster.

    PubMed

    Wang, Li; Di, Li-Jun; Lv, Xiang; Zheng, Wei; Xue, Zheng; Guo, Zhi-Chen; Liu, De-Pei; Liang, Chi-Chuan

    2009-01-01

    Matrix attachment regions (MARs) are important in chromatin organization and gene regulation. Although it is known that there are a number of MAR elements in the beta-globin gene cluster, it is unclear that how these MAR elements are involved in regulating beta-globin genes expression. Here, we report the identification of a new MAR element at the LCR (locus control region) of human beta-globin gene cluster and the detection of the inter-MAR association within the beta-globin gene cluster. Also, we demonstrate that SATB1, a protein factor that has been implicated in the formation of network like higher order chromatin structures at some gene loci, takes part in beta-globin specific inter-MAR association through binding the specific MARs. Knocking down of SATB1 obviously reduces the binding of SATB1 to the MARs and diminishes the frequency of the inter-MAR association. As a result, the ACH establishment and the alpha-like globin genes and beta-like globin genes expressions are affected either. In summary, our results suggest that SATB1 is a regulatory factor of hemoglobin genes, especially the early differentiation genes at least through affecting the higher order chromatin structure.

  13. Rad51 activates polyomavirus JC early transcription.

    PubMed

    White, Martyn K; Kaminski, Rafal; Khalili, Kamel; Wollebo, Hassen S

    2014-01-01

    The human neurotropic polyomavirus JC (JCV) causes the fatal CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection is very common and after primary infection, the virus is able to persist in an asymptomatic state. Rarely, and usually only under conditions of immune impairment, JCV re-emerges to actively replicate in the astrocytes and oligodendrocytes of the brain causing PML. The regulatory events involved in the reactivation of active viral replication in PML are not well understood but previous studies have implicated the transcription factor NF-κB acting at a well-characterized site in the JCV noncoding control region (NCCR). NF-κB in turn is regulated in a number of ways including activation by cytokines such as TNF-α, interactions with other transcription factors and epigenetic events involving protein acetylation--all of which can regulate the transcriptional activity of JCV. Active JCV infection is marked by the occurrence of rapid and extensive DNA damage in the host cell and the induction of the expression of cellular proteins involved in DNA repair including Rad51, a major component of the homologous recombination-directed double-strand break DNA repair machinery. Here we show that increased Rad51 expression activates the JCV early promoter. This activation is co-operative with the stimulation caused by NF-κB p65, abrogated by mutation of the NF-κB binding site or siRNA to NFκB p65 and enhanced by the histone deacetylase inhibitor sodium butyrate. These data indicate that the induction of Rad51 resulting from infection with JCV acts through NF-κB via its binding site to stimulate JCV early transcription. We suggest that this provides a novel positive feedback mechanism to enhance viral gene expression during the early stage of JCV infection.

  14. bldA-dependent expression of the Streptomyces exfoliatus M11 lipase gene (lipA) is mediated by the product of a contiguous gene, lipR, encoding a putative transcriptional activator.

    PubMed Central

    Servín-González, L; Castro, C; Pérez, C; Rubio, M; Valdez, F

    1997-01-01

    Extracellular lipase synthesis by Streptomyces lividans 66 carrying the cloned lipase gene (lipA) from Streptomyces exfoliatus M11 was found to be growth phase dependent, since lipase was secreted into the medium mainly during the stationary phase; S1 nuclease protection experiments revealed abundant lipA transcripts in RNA preparations obtained during the stationary phase but not in those obtained during exponential growth. Transcription from the lipA promoter was dependent on the presence of lipR, a contiguous downstream gene with a very high guanine-plus-cytosine content (80.2%). The deduced lipR product consists of a protein of 934 amino acids that shows similarity to known transcriptional activators and has a strong helix-turn-helix motif at its C terminus; this motif is part of a domain homologous to DNA-binding domains of bacterial regulators of the UhpA/LuxR superfamily. The lipR sequence revealed the presence of a leucine residue, encoded by the rare TTA codon, which caused bldA dependence of lipA transcription in Streptomyces coelicolor A3(2); replacement of the TTA codon by the alternate CTC leucine codon alleviated bidA dependence but not the apparent growth phase-dependent regulation of lipA transcription. When lipR expression was induced in a controlled fashion during the exponential growth phase, by placing it under the inducible tipA promoter, lipase synthesis was shifted to the exponential growth phase, indicating that the timing of lipR expression, and not its bldA dependence, is the main cause for stationary-phase transcription of lipA. PMID:9401043

  15. Triiodothyronine (T3) induces proinsulin gene expression by activating PI3K: possible roles for GSK-3β and the transcriptional factor PDX-1.

    PubMed

    Goulart-Silva, F; Serrano-Nascimento, C; Texeira, S S; Nunes, M T

    2013-01-01

    Thyroid hormone (TH) activates PI3K and Akt, leading to glucose uptake in rat skeletal muscle cells and proliferation of insulinoma cells, respectively. However, TH actions on pancreatic beta cells have been little explored, which lead us to evaluate the TH eff ects on proinsulin gene expression, and the involvement of PI3K/Akt/GSK-3β signaling pathway, and a transcriptional factor for insulin (PDX-1). INS-1E cells were sorted into 3 groups: control and TH-depleted treated or not with T3 for 30 min. Cells were also previously treated with actinomycin D (ActD), cycloheximide (CHX), wortmannin or Akt inhibitor. Proinsulin mRNA expression was evaluated by real time PCR, and pGSK-3β and PDX-1 protein content was analyzed by Western blotting. TH depletion decreased proinsulin mRNA content, which was restored after acute T3 treatment. ActD, CHX and wortmannin, but not Akt inhibitor, prevented the rapid stimulatory eff ect of T3 on proinsulin mRNA expression. TH depletion did not affect the phosphorylated GSK-3β and PDX-1 protein content; but T3 treatment led to an increase in the content of these proteins. These data indicate that T3 acutely increases proinsulin mRNA expression, by mechanisms which depends on the activation of PI3K, but not of Akt, and may involve the inactivation of GSK-3β by phosphorylation. Since GSK-3β enhances PDX-1 degradation rate, the GSK-3β inactivation could explain the increase of PDX-1 content in T3-treated cells. Considering that PDX-1 is one of the most important transcriptional factors for proinsulin gene expression, its enhancement may underlie the increased proinsulin mRNA content acutely induced by T3.

  16. Transcriptional regulation of the novobiocin biosynthetic gene cluster.

    PubMed

    Dangel, Volker; Härle, Johannes; Goerke, Christiane; Wolz, Christiane; Gust, Bertolt; Pernodet, Jean-Luc; Heide, Lutz

    2009-12-01

    The aminocoumarin antibiotic novobiocin is a gyrase inhibitor formed by a Streptomyces strain. The biosynthetic gene cluster of novobiocin spans 23.4 kb and contains 20 coding sequences, among them the two regulatory genes novE and novG. We investigated the location of transcriptional promoters within this cluster by insertion of transcriptional terminator cassettes and RT-PCR analysis of the resulting mutants. The cluster was found to contain eight DNA regions with promoter activity. The regulatory protein NovG binds to a previously identified binding site within the promoter region located upstream of novH, but apparently not to any of the other seven promoters. Quantitative real-time PCR was used to compare the number of transcripts in a strain carrying an intact novobiocin cluster with strains carrying mutated clusters. Both in-frame deletion of the regulatory gene novG and insertion of a terminator cassette into the biosynthetic gene novH led to a strong reduction of the number of transcripts of the genes located between novH and novW. This suggested that these 16 biosynthetic genes form a single operon. Three internal promoters are located within this operon but appear to be of minor importance, if any, under our experimental conditions. Transcription of novG was found to depend on the presence of NovE, suggesting that the two regulatory genes, novE and novG, act in a cascade-like mechanism. The resistance gene gyrB(R), encoding an aminocoumarin-resistant gyrase B subunit, may initially be co-transcribed with the genes from novH to novW. However, when the gyrase inhibitor novobiocin accumulates in the cultures, gyrB(R) is transcribed from its own promoter. Previous work has suggested that this promoter is controlled by the superhelical density of chromosomal DNA.

  17. Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.

    PubMed Central

    Blank, T E; Woods, M P; Lebo, C M; Xin, P; Hopper, J E

    1997-01-01

    Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods. PMID:9111326

  18. Thermodynamics-based models of transcriptional regulation with gene sequence.

    PubMed

    Wang, Shuqiang; Shen, Yanyan; Hu, Jinxing

    2015-12-01

    Quantitative models of gene regulatory activity have the potential to improve our mechanistic understanding of transcriptional regulation. However, the few models available today have been based on simplistic assumptions about the sequences being modeled or heuristic approximations of the underlying regulatory mechanisms. In this work, we have developed a thermodynamics-based model to predict gene expression driven by any DNA sequence. The proposed model relies on a continuous time, differential equation description of transcriptional dynamics. The sequence features of the promoter are exploited to derive the binding affinity which is derived based on statistical molecular thermodynamics. Experimental results show that the proposed model can effectively identify the activity levels of transcription factors and the regulatory parameters. Comparing with the previous models, the proposed model can reveal more biological sense.

  19. Viral double-stranded RNAs from vaccinia virus early or intermediate gene transcripts possess PKR activating function, resulting in NF-kappaB activation, when the K1 protein is absent or mutated.

    PubMed

    Willis, Kristen L; Langland, Jeffrey O; Shisler, Joanna L

    2011-03-11

    PKR is a potent antiviral molecule that can terminate infection by inhibiting protein synthesis and stimulating NF-κB activation and apoptosis. Originally, it was thought that only intermediate and late gene transcription produced double-stranded (ds) RNA to activate PKR during vaccinia virus (VACV) infection. The VACV E3 or K3 proteins squelch this effect by binding to either dsRNA or PKR. However, in the absence of the K1 protein, VACV infection activates PKR at very early times post-infection and despite the presence of E3 and K3. These data suggest that VACV infection induces PKR activation by a currently unknown mechanism. To determine this mechanism, cells were infected with K1L-containing or -deficient VACVs. By using conditions that limited the progression of the poxvirus replication cycle, we observed that early gene transcripts activated PKR in RK13 cells, identifying a new PKR-activating mechanism of poxvirus infection. Using a similar approach for HeLa cells, intermediate gene transcription was sufficient to activate PKR. RNA isolated from infected RK13 or HeLa cells maintained PKR-activating properties only when dsRNA was present. Moreover, viral dsRNA was directly detected in infected cells either by RT-PCR or immunofluorescent microscopy. Interestingly, dsRNA levels were higher in infected cells in which the K1 protein was nonfunctional. Only K1 proteins with PKR inhibitory function prevented downstream NF-κB activation. These results reveal a new PKR activation pathway during VACV infection, in which the K1 protein reduces dsRNA levels early in VACV infection to directly inhibit PKR and several of its downstream antiviral effects, thereby enhancing virus survival.

  20. Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats.

    PubMed

    Liška, František; Peterková, Renata; Peterka, Miroslav; Landa, Vladimír; Zídek, Václav; Mlejnek, Petr; Šilhavý, Jan; Šimáková, Miroslava; Křen, Vladimír; Starker, Colby G; Voytas, Daniel F; Izsvák, Zsuzsanna; Pravenec, Michal

    2016-01-01

    Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.

  1. Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats

    PubMed Central

    Liška, František; Peterková, Renata; Peterka, Miroslav; Landa, Vladimír; Zídek, Václav; Mlejnek, Petr; Šilhavý, Jan; Šimáková, Miroslava; Křen, Vladimír; Starker, Colby G.; Voytas, Daniel F.; Izsvák, Zsuzsanna; Pravenec, Michal

    2016-01-01

    Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body—column vertebrae, hindlimbs and urinary system in the rat. PMID:27727328

  2. DNA demethylation and histone H3K9 acetylation determine the active transcription of the NKG2D gene in human CD8+ T and NK cells

    PubMed Central

    Fernández-Sánchez, Alba; Baragaño Raneros, Aroa; Carvajal Palao, Reyes; Sanz, Ana B.; Ortiz, Alberto; Ortega, Francisco; Suárez-Álvarez, Beatriz; López-Larrea, Carlos

    2013-01-01

    The human activating receptor NKG2D is mainly expressed by NK, NKT, γδ T and CD8+ T cells and, under certain conditions, by CD4+ T cells. This receptor recognizes a diverse family of ligands (MICA, MICB and ULBPs 1–6) leading to the activation of effector cells and triggering the lysis of target cells. The NKG2D receptor-ligand system plays an important role in the immune response to infections, tumors, transplanted graft and autoantigens. Elucidation of the regulatory mechanisms of NKG2D is therefore essential for therapeutic purposes. In this study, we speculate whether epigenetic mechanisms, such as DNA methylation and histone acetylation, participate in NKG2D gene regulation in T lymphocytes and NK cells. DNA methylation in the NKG2D gene was observed in CD4+ T lymphocytes and T cell lines (Jurkat and HUT78), while this gene was unmethylated in NKG2D-positive cells (CD8+ T lymphocytes, NK cells and NKL cell line) and associated with high levels of histone H3 lysine 9 acetylation (H3K9Ac). Treatment with the histone acetyltransferase (HAT) inhibitor curcumin reduces H3K9Ac levels in the NKG2D gene, downregulates NKG2D transcription and leads to a marked reduction in the lytic capacity of NKG2D-mediated NKL cells. These findings suggest that differential NKG2D expression in the different cell subsets is regulated by epigenetic mechanisms and that its modulation by epigenetic treatments might provide a new strategy for treating several pathologies. PMID:23235109

  3. Src subfamily kinases regulate nuclear export and degradation of transcription factor Nrf2 to switch off Nrf2-mediated antioxidant activation of cytoprotective gene expression.

    PubMed

    Niture, Suryakant K; Jain, Abhinav K; Shelton, Phillip M; Jaiswal, Anil K

    2011-08-19

    Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3β acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis.

  4. β-Catenin and peroxisome proliferator-activated receptor-δ coordinate dynamic chromatin loops for the transcription of vascular endothelial growth factor A gene in colon cancer cells.

    PubMed

    Hwang, Injoo; Kim, Jeeho; Jeong, Sunjoo

    2012-11-30

    Vascular endothelial growth factor A (VEGFA) mRNA is regulated by β-catenin and peroxisome proliferator activated receptor δ (PPAR-δ) activation in colon cancer cells, but the detailed mechanism remains to be elucidated. As chromatin loops are generally hubs for transcription factors, we tested here whether β-catenin could modulate chromatin looping near the VEGFA gene and play any important role for PPAR-δ activated VEGFA transcription. First, we identified the far upstream site as an important site for VEGFA transcription by luciferase assay and chromatin immunoprecipitation in colorectal carcinoma HCT116 cells. Chromatin conformation capture analysis also revealed the chromatin loops formed by the β-catenin bindings on these sites near the VEGFA gene. Dynamic association and dissociation of β-catenin/TCF-4/PPAR-δ on the far upstream site and β-catenin/NF-κB p65 on the downstream site were also detected depending on PPAR-δ activation. Interestingly, β-catenin-mediated chromatin loops were relieved by PPAR-δ activation, suggesting a regulatory role of β-catenin for VEGFA transcription. Based on these data, we propose a model for PPAR-δ-activated VEGFA transcription that relies on β-catenin-mediated chromatin looping as a prerequisite for the activation. Our findings could extend to other β-catenin regulated target genes and could provide a general mechanism and novel paradigm for β-catenin-mediated oncogenesis.

  5. Osmostress-induced gene expression--a model to understand how stress-activated protein kinases (SAPKs) regulate transcription.

    PubMed

    de Nadal, Eulàlia; Posas, Francesc

    2015-09-01

    Adaptation is essential for maximizing cell survival and for cell fitness in response to sudden changes in the environment. Several aspects of cell physiology change during adaptation. Major changes in gene expression are associated with cell exposure to environmental changes, and several aspects of mRNA biogenesis appear to be targeted by signaling pathways upon stress. Exhaustive reviews have been written regarding adaptation to stress and regulation of gene expression. In this review, using osmostress in yeast as a prototypical case study, we highlight those aspects of regulation of gene induction that are general to various environmental stresses as well as mechanistic aspects that are potentially conserved from yeast to mammals.

  6. Tumor necrosis factor alpha activates transcription of the NADPH oxidase organizer 1 (NOXO1) gene and upregulates superoxide production in colon epithelial cells.

    PubMed

    Kuwano, Yuki; Tominaga, Kumiko; Kawahara, Tsukasa; Sasaki, Hidekazu; Takeo, Keiko; Nishida, Kensei; Masuda, Kiyoshi; Kawai, Tomoko; Teshima-Kondo, Shigetada; Rokutan, Kazuhito

    2008-12-15

    NADPH oxidase 1 (Nox1) is a multicomponent enzyme consisting of p22(phox), Nox organizer 1 (NOXO1), Nox1 activator 1, and Rac1. Interleukin-1beta, flagellin, interferon-gamma, and tumor necrosis factor alpha (TNF-alpha) similarly induced Nox1 in a colon cancer cell line (T84), whereas only TNF-alpha fully induced NOXO1 and upregulated superoxide-producing activity by ninefold. This upregulation was canceled by knockdown of NOXO1 with small interfering RNAs. TNF-alpha rapidly phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase 1/2, followed by phosphorylation of c-Jun and c-Fos and appearance of an AP-1 binding activity within 30 min. We cloned the 5' flank of the human NOXO1 gene (-3888 to +263 bp), and found that the region between -585 and -452 bp, which contains consensus elements of YY-1, AP-1, and Ets, and the GC-rich region encoding three putative binding sites for SP-1, was crucial for TNF-alpha-dependent promoter activity. Serial mutation analysis of the elements identified an AP-1 binding site (from -561 to -551 bp, agtAAGtcatg) as a crucial element for TNF-alpha-stimulated transcription of the human NOXO1 gene, which was also confirmed by the AP-1 decoy experiments. Thus, TNF-alpha acts as a potent activator of Nox1-based oxidase in colon epithelial cells, suggesting a potential role of this oxidase in inflammation of the colon.

  7. Promoter-proximal polyadenylation sites reduce transcription activity

    PubMed Central

    Andersen, Pia K.; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels. PMID:23028143

  8. BRCA1 transcriptionally regulates genes involved in breast tumorigenesis

    PubMed Central

    Welcsh, Piri L.; Lee, Ming K.; Gonzalez-Hernandez, Rachel M.; Black, Daniel J.; Mahadevappa, Mamatha; Swisher, Elizabeth M.; Warrington, Janet A.; King, Mary-Claire

    2002-01-01

    Loss of function of BRCA1 caused by inherited mutation and tissue-specific somatic mutation leads to breast and ovarian cancer. Nearly all BRCA1 germ-line mutations involve truncation or loss of the C-terminal BRCT transcriptional activation domain, suggesting that transcriptional regulation is a critical function of the wild-type gene. The purpose of this project was to determine whether there is a link between the role of BRCA1 in transcriptional regulation and its role in tumor suppression. We developed a cell line (in which BRCA1 can be induced) and used microarray analysis to compare transcription profiles of epithelial cells with low endogenous levels of BRCA1 vs. transcription profiles of cells with 2–4-fold higher induced levels of expression of BRCA1. At these levels of expression, BRCA1 did not induce apoptosis. Undirected cluster analysis of six paired experiments revealed 373 genes, the expression of which was altered significantly and consistently by BRCA1 induction. Expression of 62 genes was altered more than 2-fold. BRCA1-regulated genes associated with breast tumorigenesis included the estrogen-responsive genes MYC and cyclin D1, which are overexpressed in many breast tumors; STAT1 and JAK1, key components of the cytokine signal transduction pathway; the extracellular matrix protein laminin 3A; ID4, an inhibitor of DNA-binding transcriptional activators, which in turn negatively regulates BRCA1 expression; and the prohormone stanniocalcin, expression of which is lost in breast tumor cells. Coordinated expression of BRCA1 with ID4 and with stanniocalcin was confirmed in primary breast and ovarian tumors. PMID:12032322

  9. Dynamic gene expression patterns in animal models of early and late heart failure reveal biphasic-bidirectional transcriptional activation of signaling pathways.

    PubMed

    Rowell, Janelle; Koitabashi, Norimichi; Kass, David A; Barth, Andreas S

    2014-10-15

    Altered cardiac gene expression in heart failure (HF) has mostly been identified by single-point analysis of end-stage disease. This may miss earlier changes in gene expression that are transient and/or directionally opposite to those observed later. Myocardial datasets from the largest microarray data repository (Gene Expression Omnibus) yielded six HF studies with time-course data. Differentially expressed transcripts between nonfailing controls, early HF (<3 days after cardiac insult) and late HF (usually >2 wk) were determined, and analysis of KEGG pathways and predicted regulatory control elements performed. We found that gene expression followed varying patterns: Downregulation of metabolic pathways occurred early and was sustained into late-stage HF. In contrast, most signaling pathways undergo a complex biphasic pattern: Calcium signaling, p53, apoptosis, and MAPK pathways displayed a bidirectional response, declining early but rising late. These profiles were compatible with specific microRNA (miRNA) and transcription regulators: Estrogen-related receptor-α and myocyte-enhancer factor-2 binding sites were overrepresented in the promoter regions of downregulated transcripts. Concurrently, there were overrepresented binding sites for E2f and ETS family members (E-Twenty Six, including Gabp, Elf1, and Ets2), serum response and interferon regulated factor in biphasic-bidirectional and late-upregulated transcripts. Binding sites for miRNAs downregulated by HF were more common in upregulated transcripts (e.g., miRNA-22,-133a/b, and -150 in early HF and miRNA-1,-9,-499 in late HF). During the development of HF, gene expression is characterized by dynamic overlapping sets of transcripts controlled by specific interrelated regulatory mechanisms. While metabolic gene classes show early and sustained downregulation in HF, signaling pathways undergo a complex biphasic pattern with early down- and more pronounced late upregulation.

  10. A novel pax-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia.

    PubMed

    Wang, Yi-Ting; Pan, Yu-Jiao; Cho, Chao-Cheng; Lin, Bo-Chi; Su, Li-Hsin; Huang, Yu-Chang; Sun, Chin-Hung

    2010-10-15

    Giardia lamblia differentiates into infectious cysts to survive outside of the host. It is of interest to identify factors involved in up-regulation of cyst wall proteins (CWPs) during this differentiation. Pax proteins are important regulators of development and cell differentiation in Drosophila and vertebrates. No member of this gene family has been reported to date in yeast, plants, or protozoan parasites. We have identified a pax-like gene (pax1) encoding a putative paired domain in the G. lamblia genome. Epitope-tagged Pax1 localized to nuclei during both vegetative growth and encystation. Recombinant Pax1 specifically bound to the AT-rich initiator elements of the encystation-induced cwp1 to -3 and myb2 genes. Interestingly, overexpression of Pax1 increased cwp1 to -3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of the transactivation function of Pax1. Our results indicate that the Pax family has been conserved during evolution, and Pax1 could up-regulate the key encystation-induced genes to regulate differentiation of the protozoan eukaryote, G. lamblia.

  11. The Smad3 linker region contains a transcriptional activation domain.

    PubMed

    Wang, Guannan; Long, Jianyin; Matsuura, Isao; He, Dongming; Liu, Fang

    2005-02-15

    Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.

  12. NELF Potentiates Gene Transcription in the Drosophila Embryo

    PubMed Central

    Wang, Xiaoling; Hang, Saiyu; Prazak, Lisa; Gergen, J. Peter

    2010-01-01

    A hallmark of genes that are subject to developmental regulation of transcriptional elongation is association of the negative elongation factor NELF with the paused RNA polymerase complex. Here we use a combination of biochemical and genetic experiments to investigate the in vivo function of NELF in the Drosophila embryo. NELF associates with different gene promoter regions in correlation with the association of RNA polymerase II (Pol II) and the initial activation of gene expression during the early stages of embryogenesis. Genetic experiments reveal that maternally provided NELF is required for the activation, rather than the repression of reporter genes that emulate the expression of key developmental control genes. Furthermore, the relative requirement for NELF is dictated by attributes of the flanking cis-regulatory information. We propose that NELF-associated paused Pol II complexes provide a platform for high fidelity integration of the combinatorial spatial and temporal information that is central to the regulation of gene expression during animal development. PMID:20634899

  13. Gene expression and metabolite profiling of developing highbush blueberry fruit indicates transcriptional regulation of flavonoid metabolism and activation of abscisic acid metabolism.

    PubMed

    Zifkin, Michael; Jin, Alena; Ozga, Jocelyn A; Zaharia, L Irina; Schernthaner, Johann P; Gesell, Andreas; Abrams, Suzanne R; Kennedy, James A; Constabel, C Peter

    2012-01-01

    Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3'-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3'5'-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of

  14. Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism1[W][OA

    PubMed Central

    Zifkin, Michael; Jin, Alena; Ozga, Jocelyn A.; Zaharia, L. Irina; Schernthaner, Johann P.; Gesell, Andreas; Abrams, Suzanne R.; Kennedy, James A.; Constabel, C. Peter

    2012-01-01

    Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3′-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3′5′-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation

  15. Drosophilia alpha-tubulin genes and their transcription patterns.

    PubMed

    Kalfayan, L; Loewenberg, J; Wensink, P C

    1982-01-01

    There are four different alpha-tubulin genes in D. melanogaster DNA; three of them appear as single copies and the other is present as either one or two copies in the haploid genome. The transcripts of three of these genes were examined. Each of them is complementary to a transcript of different length, implying that each is transcribed. Since these transcripts are found on polysomes, it is likely that they are translated. At least two of the genes are complementary to several transcripts, indicating that each of them has more than one transcription start or stop site or perhaps that there are alternative paths of posttranscriptional processing. There is a different developmental pattern of concentrations for transcripts from each of these genes, and different RNAs from the same gene also have different patterns. We conclude that the concentration of transcripts from each gene appears to be independently controlled and that even different transcription products from the same gene appear to independently controlled.

  16. Transcriptional activation of a MYB gene controls the tissue-specific anthocyanin accumulation in a purple cauliflower mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavonoids such as anthocyanins possess significant health benefits to humans and play important physiological roles in plants. An interesting Purple gene mutation in cauliflower confers an abnormal pattern of anthocyanin accumulation, giving intense purple color in very young leaves, curds, and see...

  17. Synergistic transcriptional activation of the mouse urokinase plasminogen activator (uPA) gene and of its enhancer activator protein 1 (AP1) site by cAMP and retinoic acid.

    PubMed Central

    Mira-Y-Lopez, R; Jaramillo, S; Jing, Y

    1998-01-01

    We have investigated the mechanism whereby all-trans retinoic acid (tRA) potentiates the 8-bromo-cAMP (8-BrcAMP)-dependent transcription of the urokinase plasminogen activator (uPA) gene in SC115 mouse mammary carcinoma cells. Photoaffinity labelling experiments showed that tRA did not alter the cellular content of cAMP-dependent protein kinase regulatory subunits I and II. In agreement with this, nuclear run-on analysis in the presence of the translational inhibitor puromycin demonstrated that the effect of 8-BrcAMP and its potentiation by tRA were independent of protein synthesis. A transiently transfected 6.6 kb uPA 5'-flanking region-chloramphenicol acetyltransferase (CAT) fusion gene mimicked the response of the endogenous uPA gene. Thus 1 mM 8-BrcAMP induced a 100-200% increase in CAT content, 100 nM tRA had no effect and 100 nM tRA+1 mM 8-BrcAMP induced a 300-500% increase in cells co-transfected with tRA receptor and/or 9-cis-RA receptor. Analysis of 5'-deleted constructs showed that the tRA effect required at least two cis regions: -2657 to -2186, encompassing the 100 bp uPA enhancer, and -709 to -324, which exhibited silencing activity. Neither region contained a tRA-response element-like motif. Because tRA receptor and 9-cis-RA receptor interact with activator protein 1 (AP1), we tested whether tRA regulated the uPA enhancer AP1 site in the presence of 8-BrcAMP. We found that a dimer of this site fused to a minimal uPA-CAT fusion gene was responsive to 1 mM 8-BrcAMP (100% CAT increase), not responsive to 100 nM tRA, and synergistically responsive to 100 nM tRA+1 mM 8-BrcAMP (240% CAT increase) in cells co-transfected with Fos and Jun. Synergistic activation of the same construct and of the 6.6 kb uPA-CAT fusion gene was also obtained using tRA and 100 nM PMA. We conclude that multiple cis elements, probably including the uPA enhancer AP1 site, mediate the tRA potentiation of uPA transcription. PMID:9560322

  18. The nuclear-encoded sigma factor SIG4 directly activates transcription of chloroplast psbA and ycf17 genes in the unicellular red alga Cyanidioschyzon merolae.

    PubMed

    Fujii, Gaku; Imamura, Sousuke; Era, Atsuko; Miyagishima, Shin-ya; Hanaoka, Mitsumasa; Tanaka, Kan

    2015-05-01

    The plant organelle chloroplast originated from the endosymbiosis of a cyanobacterial-like photosynthetic bacterium, and still retains its own genome derived from this ancestor. We have been focusing on a unicellular red alga, Cyanidioschyzon merolae, as a model photosynthetic eukaryote. In this study, we analyzed the transcriptional specificity of SIG4, which is one of four nuclear-encoded chloroplast RNA polymerase sigma factors in this alga. Accumulation of the SIG4 protein was observed in response to nitrogen depletion or high light conditions. By comparing the chloroplast transcriptomes under nitrogen depletion and SIG4-overexpressing conditions, we identified several candidate genes as SIG4 targets. Together with the results of chromatin immunoprecipitation analysis, the promoters of the psbA (encoding the D1 protein of the photosystem II reaction center) and ycf17 (encoding a protein of the early light-inducible protein family) genes were shown to be direct activation targets. The phycobilisome (PBS) CpcB protein was decreased by SIG4 overexpression, which suggests the negative involvement of SIG4 in PBS accumulation.

  19. Perinatal exposure to bisphenol A modifies the transcriptional regulation of the β-Casein gene during secretory activation of the rat mammary gland.

    PubMed

    Altamirano, Gabriela A; Ramos, Jorge G; Gomez, Ayelen L; Luque, Enrique H; Muñoz-de-Toro, Monica; Kass, Laura

    2017-01-05

    With the aim to analyze whether bisphenol A (BPA) modifies β-Casein (β-Cas) synthesis and transcriptional regulation in perinatally exposed animals, here, pregnant F0 rats were orally exposed to 0, 0.6 or 52 μg BPA/kg/day from gestation day 9 until weaning. Then, F1 females were bred and mammary glands were obtained on lactation day 2. Perinatal BPA exposure decreased β-Cas expression without modifying the activation of prolactin receptor. It also decreased the expression of glucocorticoid receptor in BPA52-exposed dams and β1 and α6 integrins as well as dystroglycan in both BPA groups. In addition, BPA exposure altered the expression of histone-modifying enzymes and induced histone modifications and DNA methylation in the promoter, enhancer and exon VII of the β-Cas gene. An impaired crosstalk between the extracellular matrix and lactogenic hormone signaling pathways and epigenetic modifications of the β-Cas gene could be the molecular mechanisms by which BPA decreased β-Cas expression.

  20. The transcriptional activators AraR and XlnR from Aspergillus niger regulate expression of pentose catabolic and pentose phosphate pathway genes.

    PubMed

    Battaglia, Evy; Zhou, Miaomiao; de Vries, Ronald P

    2014-09-01

    The pentose catabolic pathway (PCP) and the pentose phosphate pathway (PPP) are required for the conversion of pentose sugars in fungi and are linked via d-xylulose-5-phosphate. Previously, it was shown that the PCP is regulated by the transcriptional activators XlnR and AraR in Aspergillus niger. Here we assessed whether XlnR and AraR also regulate the PPP. Expression of two genes, rpiA and talB, was reduced in the ΔaraR/ΔxlnR strain and increased in the xylulokinase negative strain (xkiA1) on d-xylose and/or l-arabinose. Bioinformatic analysis of the 1 kb promoter regions of rpiA and talB showed the presence of putative XlnR binding sites. Combining all results in this study, it strongly suggests that these two PPP genes are under regulation of XlnR in A. niger.

  1. TRANSCRIPTIONAL REGULATION OF THE HUMAN KiSS1 GENE

    PubMed Central

    Mueller, Johanna K.; Dietzel, Anja; Lomniczi, Alejandro; Loche, Alberto; Tefs, Katrin; Kiess, Wieland; Danne, Thomas; Ojeda, Sergio R.; Heger, Sabine

    2011-01-01

    Kisspeptin, the product of the KiSS1 gene, has emerged as a key component of the mechanism by which the hypothalamus controls puberty and reproductive development. It does so by stimulating the secretion of gonadotropin releasing hormone (GnRH). Little is known about the transcriptional control of the KiSS1 gene. Here we show that a set of proteins postulated to be upstream components of a hypothalamic network involved in controlling female puberty regulates KiSS1 transcriptional activity. Using RACE-PCR we determined that transcription of KiSS1 mRNA is initiated at a single transcription start site (TSS) located 153–156 bp upstream of the ATG translation initiation codon. Promoter assays performed using 293 MSR cells showed that the KiSS1 promoter is activated by TTF1 and CUX1-p200, and repressed by EAP1, YY1, and CUX1-p110. EAP1 and CUX-110 were also repressive in GT1-7 cells. All four TFs are recruited in vivo to the KiSS1 promoter and are expressed in kisspeptin neurons. These results suggest that expression of the KiSS1 gene is regulated by trans-activators and repressors involved in the system-wide control of mammalian puberty. PMID:21672609

  2. Cooperative antiproliferative signaling by aspirin and indole-3-carbinol targets microphthalmia-associated transcription factor gene expression and promoter activity in human melanoma cells.

    PubMed

    Poindexter, Kevin M; Matthew, Susanne; Aronchik, Ida; Firestone, Gary L

    2016-04-01

    Antiproliferative signaling of combinations of the nonsteroidal anti-inflammatory drug acetylsalicylic acid (aspirin) and indole-3-carbinol (I3C), a natural indolecarbinol compound derived from cruciferous vegetables, was investigated in human melanoma cells. Melanoma cell lines with distinct mutational profiles were sensitive to different extents to the antiproliferative response of aspirin, with oncogenic BRAF-expressing G361 cells and wild-type BRAF-expressing SK-MEL-30 cells being the most responsive. I3C triggered a strong proliferative arrest of G361 melanoma cells and caused only a modest decrease in the proliferation of SK-MEL-30 cells. In both cell lines, combinations of aspirin and I3C cooperatively arrested cell proliferation and induced a G1 cell cycle arrest, and nearly ablated protein and transcript levels of the melanocyte master regulator microphthalmia-associated transcription factor isoform M (MITF-M). In melanoma cells transfected with a -333/+120-bp MITF-M promoter-luciferase reporter plasmid, treatment with aspirin and I3C cooperatively disrupted MITF-M promoter activity, which accounted for the loss of MITF-M gene products. Mutational analysis revealed that the aspirin required the LEF1 binding site, whereas I3C required the BRN2 binding site to mediate their combined and individual effects on MITF-M promoter activity. Consistent with LEF1 being a downstream effector of Wnt signaling, aspirin, but not I3C, downregulated protein levels of the Wnt co-receptor LDL receptor-related protein-6 and β-catenin and upregulated the β-catenin destruction complex component Axin. Taken together, our results demonstrate that aspirin-regulated Wnt signaling and I3C-targeted signaling pathways converge at distinct DNA elements in the MITF-M promoter to cooperatively disrupt MITF-M expression and melanoma cell proliferation.

  3. A basic/hydrophobic cleft of the T4 activator MotA interacts with the C-terminus of E. coli σ70 to activate middle gene transcription

    PubMed Central

    Bonocora, Richard P.; Caignan, Gregori; Woodrell, Christopher; Werner, Milton H.; Hinton, Deborah M.

    2008-01-01

    Summary Transcriptional activation often employs a direct interaction between an activator and RNA polymerase. For activation of its middle genes, bacteriophage T4 appropriates E. coli RNA polymerase through the action of two phage-encoded proteins, MotA and AsiA. Alone, AsiA inhibits transcription from a large class of host promoters by structurally remodeling region 4 of σ70, the primary specificity subunit of E. coli RNA polymerase. MotA interacts both with σ70 region 4 and with a DNA element present in T4 middle promoters. AsiA-induced remodeling is proposed to make the far C-terminus of σ70 region 4 accessible for MotA binding. Here, NMR chemical shift analysis indicates that MotA uses a “basic/hydrophobic” cleft to interact with the C-terminus of AsiA-remodeled σ70, but MotA does not interact with AsiA itself. Mutations within this cleft, at residues K3, K28, and Q76, both impair the interaction of MotA with σ70 region 4 and MotA-dependent activation. Furthermore, mutations at these residues greatly decrease phage viability. Most previously described activators that target σ70 directly use acidic residues to engage a basic surface of region 4. Our work supports accumulated evidence indicating that “σ appropriation” by MotA and AsiA uses a fundamentally different mechanism to activate transcription. PMID:18485078

  4. Regulatory xenobiotic responsive elements in the distal 5'-flanking region of the mouse Cyp1a2 gene required for transcriptional activation by 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    PubMed

    Kawasaki, Yuki; Sakuma, Tsutomu; Goto, Yuma; Nemoto, Nobuo

    2010-10-01

    We examined the xenobiotic responsive element (XRE) responsible for induction of the mouse Cyp1a2 gene by 3-methylcholanthrene (3MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) using a reporter gene assay in mouse hepatocytes in primary culture. Although, the 5'-flanking region up to -9.5 kilobase pairs did not show a significant increase in transcriptional activity after treatment with 3MC or TCDD, a further distal 5'-flanking region from -13,958 to -12,520 containing 12 putative XREs (5'-GCGTG-3') demonstrated distinctive transcriptional activity after treatment with 3MC or TCDD. When a mutation was introduced into XRE14 at -12,972, the activation was decreased, and concurrent mutations in XRE14, XRE13, and XRE15 completely abolished it. However, mutations in XRE13, XRE15, XRE16, or XRE17 did not affect the inducible transcriptional activation of the mouse Cyp1a2 gene. These results suggest that XRE14 is important and that XRE13 at -12,897 and/or XRE15 at -13,061 are cooperative to the inducible transcriptional activation of the mouse Cyp1a2 gene by ligands of the aryl hydrocarbon receptor.

  5. Specificity Protein 1 (Sp1)-dependent Activation of the Synapsin I Gene (SYN1) Is Modulated by RE1-silencing Transcription Factor (REST) and 5′-Cytosine-Phosphoguanine (CpG) Methylation*

    PubMed Central

    Paonessa, Francesco; Latifi, Shahrzad; Scarongella, Helena; Cesca, Fabrizia; Benfenati, Fabio

    2013-01-01

    The development and function of the nervous system are directly dependent on a well defined pattern of gene expression. Indeed, perturbation of transcriptional activity or epigenetic modifications of chromatin can dramatically influence neuronal phenotypes. The phosphoprotein synapsin I (Syn I) plays a crucial role during axonogenesis and synaptogenesis as well as in synaptic transmission and plasticity of mature neurons. Abnormalities in SYN1 gene expression have been linked to important neuropsychiatric disorders, such as epilepsy and autism. SYN1 gene transcription is suppressed in non-neural tissues by the RE1-silencing transcription factor (REST); however, the molecular mechanisms that allow the constitutive expression of this genetic region in neurons have not been clarified yet. Herein we demonstrate that a conserved region of human and mouse SYN1 promoters contains cis-sites for the transcriptional activator Sp1 in close proximity to REST binding motifs. Through a series of functional assays, we demonstrate a physical interaction of Sp1 on the SYN1 promoter and show that REST directly inhibits Sp1-mediated transcription, resulting in SYN1 down-regulation. Upon differentiation of neuroblastoma Neuro2a cells, we observe a decrease in endogenous REST and a higher stability of Sp1 on target GC boxes, resulting in an increase of SYN1 transcription. Moreover, methylation of Sp1 cis-sites in the SYN1 promoter region could provide an additional level of transcriptional regulation. Our results introduce Sp1 as a fundamental activator of basal SYN1 gene expression, whose activity is modulated by the neural master regulator REST and CpG methylation. PMID:23250796

  6. p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene

    PubMed Central

    He, Z; Liu, H; Agostini, M; Yousefi, S; Perren, A; Tschan, M P; Mak, T W; Melino, G; Simon, H U

    2013-01-01

    p73, a member of the p53 tumor suppressor family, is involved in neurogenesis, sensory pathways, immunity, inflammation, and tumorigenesis. How p73 is able to participate in such a broad spectrum of different biological processes is still largely unknown. Here, we report a novel role of p73 in regulating lipid metabolism by direct transactivation of the promoter of autophagy-related protein 5 (ATG5), a gene whose product is required for autophagosome formation. Following nutrient deprivation, the livers of p73-deficient mice demonstrate a massive accumulation of lipid droplets, together with a low level of autophagy, suggesting that triglyceride hydrolysis into fatty acids is blocked owing to deficient autophagy (macrolipophagy). Compared with wild-type mice, mice functionally deficient in all the p73 isoforms exhibit decreased ATG5 expression and lower levels of autophagy in multiple organs. We further show that the TAp73α is the critical p73 isoform responsible for inducing ATG5 expression in a p53-independent manner and demonstrate that ATG5 gene transfer can correct autophagy and macrolipophagy defects in p73-deficient hepatocytes. These data strongly suggest that the p73–ATG5 axis represents a novel, key pathway for regulating lipid metabolism through autophagy. The identification of p73 as a major regulator of autophagy suggests that it may have an important role in preventing or delaying disease and aging by maintaining a homeostatic control. PMID:23912709

  7. p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene.

    PubMed

    He, Z; Liu, H; Agostini, M; Yousefi, S; Perren, A; Tschan, M P; Mak, T W; Melino, G; Simon, H U

    2013-10-01

    p73, a member of the p53 tumor suppressor family, is involved in neurogenesis, sensory pathways, immunity, inflammation, and tumorigenesis. How p73 is able to participate in such a broad spectrum of different biological processes is still largely unknown. Here, we report a novel role of p73 in regulating lipid metabolism by direct transactivation of the promoter of autophagy-related protein 5 (ATG5), a gene whose product is required for autophagosome formation. Following nutrient deprivation, the livers of p73-deficient mice demonstrate a massive accumulation of lipid droplets, together with a low level of autophagy, suggesting that triglyceride hydrolysis into fatty acids is blocked owing to deficient autophagy (macrolipophagy). Compared with wild-type mice, mice functionally deficient in all the p73 isoforms exhibit decreased ATG5 expression and lower levels of autophagy in multiple organs. We further show that the TAp73α is the critical p73 isoform responsible for inducing ATG5 expression in a p53-independent manner and demonstrate that ATG5 gene transfer can correct autophagy and macrolipophagy defects in p73-deficient hepatocytes. These data strongly suggest that the p73-ATG5 axis represents a novel, key pathway for regulating lipid metabolism through autophagy. The identification of p73 as a major regulator of autophagy suggests that it may have an important role in preventing or delaying disease and aging by maintaining a homeostatic control.

  8. The transcriptional regulation of the human CYP2C genes

    PubMed Central

    Chen, Yuping; Goldstein, Joyce A.

    2010-01-01

    In humans, four members of the CYP2C subfamily (CYP2C8, CYP2C9, CYP2C18, and CYP2C19) metabolize more than 20% of all therapeutic drugs as well as a number of endogenous compounds. The CYP2C enzymes are found predominantly in the liver, where they comprise ∼20% of the total cytochrome P450. A variety of xenobiotics such as phenobarbital, rifampicin, and hyperforin have been shown to induce the transcriptional expression of CYP2C genes in primary human hepatocytes and to increase the metabolism of CYP2C substrates in vivo in man. This induction can result in drug-drug interactions, drug tolerance, and therapeutic failure. Several drug-activated nuclear receptors including CAR, PXR, VDR, and GR recognize drug responsive elements within the 5′ flanking promoter region of CYP2C genes to mediate the transcriptional upregulation of these genes in response to xenobiotics and steroids. Other nuclear receptors and transcriptional factors including HNF4α, HNF3γ, C/EBPα and more recently RORs, have been reported to regulate the constitutive expression of CYP2C genes in liver. The maximum transcriptional induction of CYP2C genes appears to be achieved through a coordinative cross-talk between drug responsive nuclear receptors, hepatic factors, and coactivators. The transcriptional regulatory mechanisms of the expression of CYP2C genes in extrahepatic tissues has received less study, but these may be altered by perturbations from pathological conditions such as ischemia as well as some of the receptors mentioned above. PMID:19702536

  9. Gene Isoform Specificity through Enhancer-Associated Antisense Transcription

    PubMed Central

    Onodera, Courtney S.; Underwood, Jason G.; Katzman, Sol; Jacobs, Frank; Greenberg, David; Salama, Sofie R.; Haussler, David

    2012-01-01

    Enhancers and antisense RNAs play key roles in transcriptional regulation through differing mechanisms. Recent studies have demonstrated that enhancers are often associated with non-coding RNAs (ncRNAs), yet the functional role of these enhancer:ncRNA associations is unclear. Using RNA-Sequencing to interrogate the transcriptomes of undifferentiated mouse embryonic stem cells (mESCs) and their derived neural precursor cells (NPs), we identified two novel enhancer-associated antisense transcripts that appear to control isoform-specific expression of their overlapping protein-coding genes. In each case, an enhancer internal to a protein-coding gene drives an antisense RNA in mESCs but not in NPs. Expression of the antisense RNA is correlated with expression of a shorter isoform of the associated sense gene that is not present when the antisense RNA is not expressed. We demonstrate that expression of the antisense transcripts as well as expression of the short sense isoforms correlates with enhancer activity at these two loci. Further, overexpression and knockdown experiments suggest the antisense transcripts regulate expression of their associated sense genes via cis-acting mechanisms. Interestingly, the protein-coding genes involved in these two examples, Zmynd8 and Brd1, share many functional domains, yet their antisense ncRNAs show no homology to each other and are not present in non-murine mammalian lineages, such as the primate lineage. The lack of homology in the antisense ncRNAs indicates they have evolved independently of each other and suggests that this mode of lineage-specific transcriptional regulation may be more widespread in other cell types and organisms. Our findings present a new view of enhancer action wherein enhancers may direct isoform-specific expression of genes through ncRNA intermediates. PMID:22937057

  10. Inferring transcription factor collaborations in gene regulatory networks

    PubMed Central

    2014-01-01

    Background Living cells are realized by complex gene expression programs that are moderated by regulatory proteins called transcription factors (TFs). The TFs control the differential expression of target genes in the context of transcriptional regulatory networks (TRNs), either individually or in groups. Deciphering the mechanisms of how the TFs control the expression of target genes is a challenging task, especially when multiple TFs collaboratively participate in the transcriptional regulation. Results We model the underlying regulatory interactions in terms of the directions (activation or repression) and their logical roles (necessary and/or sufficient) with a modified association rule mining approach, called mTRIM. The experiment on Yeast discovered 670 regulatory interactions, in which multiple TFs express their functions on common target genes collaboratively. The evaluation on yeast genetic interactions, TF knockouts and a synthetic dataset shows that our algorithm is significantly better than the existing ones. Conclusions mTRIM is a novel method to infer TF collaborations in transcriptional regulation networks. mTRIM is available at http://www.msu.edu/~jinchen/mTRIM. PMID:24565025

  11. Proteolytic activation of pro-spätzle is required for the induced transcription of antimicrobial peptide genes in lepidopteran insects

    PubMed Central

    Wang, Yang; Cheng, Tingcai; Rayaprolu, Subrahmanyam; Zou, Zhen; Xia, Qingyou; Xiang, Zhonghuai; Jiang, Haobo

    2007-01-01

    Microbial infection leads to proteolytic activation of Drosophila spätzle, which binds to the toll receptor and induces the synthesis of immune proteins. To test whether or not this mechanism exists in lepidopteran insects, we cloned the cDNA of Bombyx mori spätzle-1 and overexpressed the full-length and truncated BmSpz1 cDNA in Escherichia coli. The insoluble fusion proteins were affinity-purified under denaturing condition. After the silkworm larvae were injected with renatured BmSpz1, mRNA levels of antimicrobial peptide genes greatly increased. Similar transcriptional up-regulation was also found in Manduca sexta. Injection of pro-BmSpz1 had no such effect. When pro-BmSpz1 and Micrococcus luteus were incubated with the plasma from M. sexta larvae, we detected proteolytic processing of pro-BmSpz1. These results suggest that active spätzle is required for the induced production of antimicrobial peptides in B. mori and M. sexta. PMID:17337053

  12. The Heterochromatic Rolled Gene of Drosophila Melanogaster Is Extensively Polytenized and Transcriptionally Active in the Salivary Gland Chromocenter

    PubMed Central

    Berghella, L.; Dimitri, P.

    1996-01-01

    This paper reports a cytogenetic and molecular study of the structural and functional organization of the Drosophila melanogaster chromocenter. The relations between mitotic (constitutive) heterochromatin and α- and β-heterochromatin are not fully understood. In the present work, we have studied the polytenization of the rolled (rl) locus, a 100-kb genomic region that maps to the proximal heterochromatin of chromosome 2 and has been previously thought to contribute to α-heterochromatin. We show that rolled undergoes polytenization in salivary gland chromosomes to a degree comparable to that of euchromatic genes, despite its deep heterochromatic location. In contrast, both the Bari-1 sequences and the AAGAC satellite repeats, located respectively to the left and right of rl, are severely underrepresented and thus both appear to be α-heterochromatic. In addition, we found that rl is transcribed in polytene tissues. Together, the results reported here indicate that functional sequences located within the proximal constitutive heterochromatin can undergo polytenization, contributing to the formation of β-heterochromatin. The implications of this finding to chromocenter structure are discussed. PMID:8878678

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

    PubMed Central

    Hahn, Steven; Young, Elton T.

    2011-01-01

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

  14. Transcriptional regulation of steroid hydroxylase genes by corticotropin.

    PubMed Central

    John, M E; John, M C; Boggaram, V; Simpson, E R; Waterman, M R

    1986-01-01

    Maintenance of optimal steroidogenic capacity in the adrenal cortex is the result of a cAMP-dependent response to the peptide hormone corticotropin (ACTH). The molecular mechanism of this action of ACTH has been examined by using five recombinant DNA clones specific for enzymes of the steroidogenic pathway (P-450scc, P-45011 beta, P-450C21, P-45017 alpha, and adrenodoxin). The presence of nuclear precursors in steady-state RNA samples derived from cultured bovine adrenocortical cells and moderate increases in the number of RNA chain initiations, as determined by in vitro nuclear run-off assays, indicate that ACTH controls the expression of the gene(s) for each of these proteins at the transcriptional level. The ACTH-mediated increase in accumulation of transcripts specific for steroid hydroxylases in nuclear RNA can be specifically blocked by inhibiting protein synthesis in bovine adrenocortical cell cultures. The steady-state concentrations of nuclear RNA for control genes show no decrease upon cycloheximide treatment. These studies suggest that a primary action of ACTH in the adrenal cortex is to activate (via cAMP) the synthesis of rapidly turning over protein factors that in turn mediate increased initiation of transcription of steroid hydroxylase genes. We propose that these protein factors impart specificity of induction to genes encoding components of this pathway in steroidogenic tissues. Images PMID:3014507

  15. Transcriptional regulation of Bacillus subtilis citrate synthase genes.

    PubMed

    Jin, S; Sonenshein, A L

    1994-08-01

    The Bacillus subtilis citrate synthase genes citA and citZ were repressed during early exponential growth phase in nutrient broth medium and were induced as cells reached the end of exponential phase. Both genes were also induced by treatment of cells with the drug decoyinine. After induction, the steady-state level of citZ mRNA was about five times higher than that of citA mRNA. At least some of the citZ transcripts read through into the isocitrate dehydrogenase (citC) gene. Transcription from an apparent promoter site located near the 3' end of the citZ gene also contributed to expression of citC. In minimal medium, citA transcription was about 6-fold lower when glucose was the sole carbon source than it was when succinate was the carbon source. Expression of the citZ gene was repressed 2-fold by glucose and 10-fold when glucose and glutamate were present simultaneously. This latter synergistic repression is similar to the effect of glucose and glutamate on steady-state citrate synthase enzyme activity. CitR, a protein of the LysR family, appeared to be a repressor of citA but not of citZ.

  16. The involvement of Opaque 2 on beta-prolamin gene regulation in maize and Coix suggests a more general role for this transcriptional activator.

    PubMed

    Cord Neto, G; Yunes, J A; da Silva, M J; Vettore, A L; Arruda, P; Leite, A

    1995-03-01

    The maize opaque 2 (o2) mutation is known to have numerous pleiotropic effects. Some polypeptides have their expression depressed while others are enhanced. The best characterized effects of the o2 mutation are those exerted on endosperm genes encoding the storage protein class of the 22 kDa alpha-zeins and the ribosome inactivating protein b-32. The Opaque 2 (O2) locus encodes a basic domain-leucine zipper DNA-binding factor, O2, which transcriptionally regulates these genes. In the maize-related grass Coix lacryma-jobi, an O2-homologous protein regulates the 25 kDa alpha-coixin family. We show in this paper that O2 transcriptionally regulates the structurally and developmentally different class of the beta-prolamins. A new O2-binding box was identified in beta-prolamin genes from maize and Coix that, together with the boxes previously identified in other endosperm expressed genes, forms a curious collection of O2 cis elements. This may have regulatory implications on the role of O2 in the mechanism that controls coordinated gene expression in the developing endosperm. Considering that the O2 locus controls at least three distinct classes of genes in maize endosperm, we propose that the O2 protein may play a more general role in maize endosperm development than previously conceived.

  17. Transcription-coupled changes to chromatin underpin gene silencing by transcriptional interference.

    PubMed

    Ard, Ryan; Allshire, Robin C

    2016-12-15

    Long non-coding RNA (lncRNA) transcription into a downstream promoter frequently results in transcriptional interference. However, the mechanism of this repression is not fully understood. We recently showed that drug tolerance in fission yeast Schizosaccharomyces pombe is controlled by lncRNA transcription upstream of the tgp1(+) permease gene. Here we demonstrate that transcriptional interference of tgp1(+) involves several transcription-coupled chromatin changes mediated by conserved elongation factors Set2, Clr6CII, Spt6 and FACT. These factors are known to travel with RNAPII and establish repressive chromatin in order to limit aberrant transcription initiation from cryptic promoters present in gene bodies. We therefore conclude that conserved RNAPII-associated mechanisms exist to both suppress intragenic cryptic promoters during genic transcription and to repress gene promoters by transcriptional interference. Our analyses also demonstrate that key mechanistic features of transcriptional interference are shared between S. pombe and the highly divergent budding yeast Saccharomyces cerevisiae Thus, transcriptional interference is an ancient, conserved mechanism for tightly controlling gene expression. Our mechanistic insights allowed us to predict and validate a second example of transcriptional interference involving the S. pombe pho1(+) gene. Given that eukaryotic genomes are pervasively transcribed, transcriptional interference likely represents a more general feature of gene regulation than is currently appreciated.

  18. NADPH oxidase 4 regulates cardiomyocyte differentiation via redox activation of c-Jun protein and the cis-regulation of GATA-4 gene transcription.

    PubMed

    Murray, Thomas V A; Smyrnias, Ioannis; Shah, Ajay M; Brewer, Alison C

    2013-05-31

    NADPH oxidase 4 (Nox4) generates reactive oxygen species (ROS) that can modulate cellular phenotype and function in part through the redox modulation of the activity of transcription factors. We demonstrate here the potential of Nox4 to drive cardiomyocyte differentiation in pluripotent embryonal carcinoma cells, and we show that this involves the redox activation of c-Jun. This in turn acts to up-regulate GATA-4 expression, one of the earliest markers of cardiotypic differentiation, through a defined and highly conserved cis-acting motif within the GATA-4 promoter. These data therefore suggest a mechanism whereby ROS act in pluripotential cells in vivo to regulate the initial transcription of critical tissue-restricted determinant(s) of the cardiomyocyte phenotype, including GATA-4. The ROS-dependent activation, mediated by Nox4, of widely expressed redox-regulated transcription factors, such as c-Jun, is fundamental to this process.

  19. Characterization of four heat-shock protein genes from Nile tilapia (Oreochromis niloticus) and demonstration of the inducible transcriptional activity of Hsp70 promoter.

    PubMed

    Zhang, Lili; Sun, Chengfei; Ye, Xing; Zou, Shuming; Lu, Maixin; Liu, Zhigang; Tian, Yuanyuan

    2014-02-01

    Heat-shock proteins (Hsps), known as stress proteins and extrinsic chaperones, play important roles in the folding, translocation, and refolding/degradation of proteins. In this study, we identified four Hsps in Nile tilapia (Oreochromis niloticus), which display conserved Hsp characteristics in their predicted amino acid sequences. Further analyses on the structures, homology, and phylogenetics revealed that the four Hsps belong to Hsp70 family. One of them does not contain introns and is named Hsp70, while all the other three contain introns and are named Hsc70-1, Hsc70-2, and Hsc70-3. Expressions of the four Hsp proteins were observed in all examined tissues. Six hours after infection of Streptococcus agalactiae in Nile tilapia, the expression of Hsp70 was significantly increased in the liver, head kidney, spleen and gill, while Hsc70s' expression was unchanged in all examined tissues except the head kidney that showed significantly reduced expression of both Hsc70-2 and Hsc70-3. These results suggest that Hsp70 may participate in the defense against S. agalactiae infection. We then isolated the promoter of Hsp70 gene and inserted it into the donor plasmid of Tgf2 transposon system containing green fluorescent protein (GFP) gene. The plasmid was microinjected into zebrafish embryos, where the expression of GFP was induced by heat shock, S. agalactiae immersion challenge, indicating that the isolated Hsp70 promoter has transcriptional activity and is inducible by both heat shock and bacterial challenge. This promoter may facilitate the future construction of disease-resistant transgenic fish. The work also contributes to the further study of immune response of tilapia after bacterial infection.

  20. Allele-specific transcriptional activity of the variable number of tandem repeats of the inducible nitric oxide synthase gene is associated with idiopathic achalasia

    PubMed Central

    Grosso, Michela; Palumbo, Ilaria; Pesce, Marcella; D’Alessandro, Alessandra; Zaninotto, Giovanni; Annese, Vito; Petruzzelli, Raffaella; Izzo, Paola; Sepulveres, Rossana; Bruzzese, Dario; Esposito, Giuseppe; Cuomo, Rosario

    2016-01-01

    Background Polymorphisms of genes involved in the regulation of the immune response are risk factors for achalasia, but their contribution to disease pathogenesis is unknown. Nitric oxide is involved both in immune function and inhibitory neurotransmission. Objective The objective of this article is to assess the association and the functional relevance of the CCTTT-inducible nitric oxide synthase (NOS2) gene promoter polymorphism in achalasia. Methods Genomic DNA was isolated from 181 achalasia patients and 220 controls. Genotyping of the (CCTTT)n repeats was performed by PCR and capillary electrophoresis, and data analyzed by considering the frequency of the different alleles. HT29 cells were transfected with iNOS luciferase promoter-reporter plasmids containing different (CCTTT)n. Results The alleles’ distribution ranged from 7 to 18, with a peak frequency at 12 repeats. Analysis of the allele frequencies revealed that individuals carrying 10 and 13 CCTTT repeats were respectively less and more frequent in achalasia (OR 0.5, 95% CI 0.3–0.5 and OR 1.6, 95% CI 1–2.4, all p < 0.05). Long repeats were also significantly associated with an earlier onset of the disease (OR 1.69, 95% CI 1.13–2.53, p = 0.01). Transfection experiments revealed a similar allele-specific iNOS transcriptional activity. Conclusion The functional polymorphism (CCTTT) of NOS2 promoter is associated with achalasia, likely by an allele-specific modulation of nitric oxide production. PMID:28344787

  1. The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium.

    PubMed

    Reyes-Olalde, J Irepan; Zúñiga-Mayo, Víctor M; Serwatowska, Joanna; Chavez Montes, Ricardo A; Lozano-Sotomayor, Paulina; Herrera-Ubaldo, Humberto; Gonzalez-Aguilera, Karla L; Ballester, Patricia; Ripoll, Juan José; Ezquer, Ignacio; Paolo, Dario; Heyl, Alexander; Colombo, Lucia; Yanofsky, Martin F; Ferrandiz, Cristina; Marsch-Martínez, Nayelli; de Folter, Stefan

    2017-04-07

    Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.

  2. Transcriptional regulation of the ferritin heavy-chain gene: the activity of the CCAAT binding factor NF-Y is modulated in heme-treated Friend leukemia cells and during monocyte-to-macrophage differentiation.

    PubMed Central

    Marziali, G; Perrotti, E; Ilari, R; Testa, U; Coccia, E M; Battistini, A

    1997-01-01

    The ferritin H-chain gene promoter regulation was analyzed in heme-treated Friend leukemia cells (FLCs) and during monocyte-to-macrophage differentiation. In the majority of cell lines studied, the regulation of ferritin expression was exerted mostly at the translational level. However, in differentiating erythroid cells, which must incorporate high levels of iron to sustain hemoglobin synthesis, and in macrophages, which are involved in iron storage, transcriptional regulation seemed to be a relevant mechanism. We show here that the minimum region of the ferritin H-gene promoter that is able to confer transcriptional regulation by heme in FLCs to a reporter gene is 77 nucleotides upstream of the TATA box. This cis element binds a protein complex referred to as HRF (heme-responsive factor), which is greatly enhanced both in heme-treated FLCs and during monocyte-to-macrophage differentiation. The CCAAT element present in reverse orientation in this promoter region of the ferritin H-chain gene is necessary for binding and for gene activity, since a single point mutation is able to abolish the binding of HRF and the transcriptional activity in transfected cells. By competition experiments and supershift assays, we identified the induced HRF as containing at least the ubiquitous transcription factor NF-Y. NF-Y is formed by three subunits, A, B, and C, all of which are necessary for DNA binding. Cotransfection with a transdominant negative mutant of the NF-YA subunit abolishes the transcriptional activation by heme, indicating that NF-Y plays an essential role in this activation. We have also observed a differential expression of the NF-YA subunit in heme-treated and control FLCs and during monocyte-to-macrophage differentiation. PMID:9032265

  3. Stochastic model for gene transcription on Drosophila melanogaster embryos

    NASA Astrophysics Data System (ADS)

    Prata, Guilherme N.; Hornos, José Eduardo M.; Ramos, Alexandre F.

    2016-02-01

    We examine immunostaining experimental data for the formation of stripe 2 of even-skipped (eve) transcripts on D. melanogaster embryos. An estimate of the factor converting immunofluorescence intensity units into molecular numbers is given. The analysis of the eve dynamics at the region of stripe 2 suggests that the promoter site of the gene has two distinct regimes: an earlier phase when it is predominantly activated until a critical time when it becomes mainly repressed. That suggests proposing a stochastic binary model for gene transcription on D. melanogaster embryos. Our model has two random variables: the transcripts number and the state of the source of mRNAs given as active or repressed. We are able to reproduce available experimental data for the average number of transcripts. An analysis of the random fluctuations on the number of eves and their consequences on the spatial precision of stripe 2 is presented. We show that the position of the anterior or posterior borders fluctuate around their average position by ˜1 % of the embryo length, which is similar to what is found experimentally. The fitting of data by such a simple model suggests that it can be useful to understand the functions of randomness during developmental processes.

  4. Transcriptional regulation of human thromboxane synthase gene expression

    SciTech Connect

    Lee, K.D.; Baek, S.J.; Fleischer, T

    1994-09-01

    The human thromboxane synthase (TS) gene encodes a microsomal enzyme catalyzing the conversion of prostaglandin endoperoxide into thromboxane A{sub 2}(TxA{sub 2}), a potent inducer of vasoconstriction and platelet aggregation. A deficiency in platelet TS activity results in bleeding disorders, but the underlying molecular mechanism remains to be elucidated. Increased TxA{sub 2} has been associated with many pathophysiological conditions such as cardiovascular disease, pulmonary hypertension, pre-eclampsia, and thrombosis in sickle cell patients. Since the formation of TxA{sub 2} is dependent upon TS, the regulation of TS gene expression may presumably play a crucial role in vivo. Abrogation of the regulatory mechanism in TS gene expression might contribute, in part, to the above clinical manifestations. To gain insight into TS gene regulation, a 1.7 kb promoter of the human TS gene was cloned and sequenced. RNase protection assay and 5{prime} RACE protocols were used to map the transcription initiation site to nucleotide A, 30 bp downstream from a canonical TATA box. Several transcription factor binding sites, including AP-1, PU.1, and PEA3, were identified within this sequence. Transient expression studies in HL-60 cells transfected with constructs containing various lengths (0.2 to 5.5 kb) of the TS promoter/luciferase fusion gene indicated the presence of multiple repressor elements within the 5.5 kb TS promoter. However, a lineage-specific up-regulation of TS gene expression was observed in HL-60 cells induced by TPA to differentiate along the macrophage lineage. The increase in TS transcription was not detectable until 36 hr after addition of the inducer. These results suggest that expression of the human TS gene may be regulated by a mechanism involving repression and derepression of the TS promoter.

  5. Regulation of maternal transcript destabilization during egg activation in Drosophila.

    PubMed Central

    Tadros, Wael; Houston, Simon A; Bashirullah, Arash; Cooperstock, Ramona L; Semotok, Jennifer L; Reed, Bruce H; Lipshitz, Howard D

    2003-01-01

    In animals, the transfer of developmental control from maternal RNAs and proteins to zygotically derived products occurs at the midblastula transition. This is accompanied by the destabilization of a subset of maternal transcripts. In Drosophila, maternal transcript destabilization occurs in the absence of fertilization and requires specific cis-acting instability elements. We show here that egg activation is necessary and sufficient to trigger transcript destabilization. We have identified 13 maternal-effect lethal loci that, when mutated, result in failure of maternal transcript degradation. All mutants identified are defective in one or more additional processes associated with egg activation. These include vitelline membrane reorganization, cortical microtubule depolymerization, translation of maternal mRNA, completion of meiosis, and chromosome condensation (the S-to-M transition) after meiosis. The least pleiotropic class of transcript destabilization mutants consists of three genes: pan gu, plutonium, and giant nuclei. These three genes regulate the S-to-M transition at the end of meiosis and are thought to be required for the maintenance of cyclin-dependent kinase (CDK) activity during this cell cycle transition. Consistent with a possible functional connection between this S-to-M transition and transcript destabilization, we show that in vitro-activated eggs, which exhibit aberrant postmeiotic chromosome condensation, fail to initiate transcript degradation. Several genetic tests exclude the possibility that reduction of CDK/cyclin complex activity per se is responsible for the failure to trigger transcript destabilization in these mutants. We propose that the trigger for transcript destabilization occurs coincidently with the S-to-M transition at the end of meiosis and that pan gu, plutonium, and giant nuclei regulate maternal transcript destabilization independent of their role in cell cycle regulation. PMID:12871909

  6. Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism

    PubMed Central

    Campos-Sandoval, José A.; Manzanares, Elisa; Lobo, Carolina; Segura, J. A.; Alonso, Francisco J.; Matés, José M.; Márquez, Javier

    2012-01-01

    Background Glutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene. Methodology/Principal Findings We demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed. Conclusions/Significance This is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was

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

    PubMed

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

    2011-03-10

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

  8. The effect of aluminium-stress and exogenous spermidine on chlorophyll degradation, glutathione reductase activity and the photosystem II D1 protein gene (psbA) transcript level in lichen Xanthoria parietina.

    PubMed

    Sen, Gulseren; Eryilmaz, Isil Ezgi; Ozakca, Dilek

    2014-02-01

    In this study, the effects of short-term aluminium toxicity and the application of spermidine on the lichen Xanthoria parietina were investigated at the physiological and transcriptional levels. Our results suggest that aluminium stress leads to physiological processes in a dose-dependent manner through differences in lipid peroxidation rate, chlorophyll content and glutathione reductase (EC 1.6.4.2) activity in aluminium and spermidine treated samples. The expression of the photosystem II D1 protein (psbA) gene was quantified using semi-quantitative RT-PCR. Increased glutathione reductase activity and psbA mRNA transcript levels were observed in the X. parietina thalli that were treated with spermidine before aluminium-stress. The results showed that the application of spermidine could mitigate aluminium-induced lipid peroxidation and chlorophyll degradation on lichen X. parietina thalli through an increase in psbA transcript levels and activity of glutathione reductase (GR) enzymes.

  9. Land use type significantly affects microbial gene transcription in soil.

    PubMed

    Nacke, Heiko; Fischer, Christiane; Thürmer, Andrea; Meinicke, Peter; Daniel, Rolf

    2014-05-01

    Soil microorganisms play an essential role in sustaining biogeochemical processes and cycling of nutrients across different land use types. To gain insights into microbial gene transcription in forest and grassland soil, we isolated mRNA from 32 sampling sites. After sequencing of generated complementary DNA (cDNA), a total of 5,824,229 sequences could be further analyzed. We were able to assign nonribosomal cDNA sequences to all three domains of life. A dominance of bacterial sequences, which were affiliated to 25 different phyla, was found. Bacterial groups capable of aromatic compound degradation such as Phenylobacterium and Burkholderia were detected in significantly higher relative abundance in forest soil than in grassland soil. Accordingly, KEGG pathway categories related to degradation of aromatic ring-containing molecules (e.g., benzoate degradation) were identified in high abundance within forest soil-derived metatranscriptomic datasets. The impact of land use type forest on community composition and activity is evidently to a high degree caused by the presence of wood breakdown products. Correspondingly, bacterial groups known to be involved in lignin degradation and containing ligninolytic genes such as Burkholderia, Bradyrhizobium, and Azospirillum exhibited increased transcriptional activity in forest soil. Higher solar radiation in grassland presumably induced increased transcription of photosynthesis-related genes within this land use type. This is in accordance with high abundance of photosynthetic organisms and plant-infecting viruses in grassland.

  10. Effects of hemorrhage on cytokine gene transcription.

    PubMed

    Shenkar, R; Abraham, E

    1993-08-01

    Injury and blood loss are often followed by infection and the rapid development of organ system dysfunction, frequently involving mucosal sites, such as the lung and intestine. To examine possible mechanisms contributing to these conditions, we used semiquantitative polymerase chain reactions to determine cytokine mRNA expression among cellular populations isolated from mucosal and systemic anatomic sites of mice at predetermined time points following 30% blood volume hemorrhage with resuscitation 1 hr later. Within 1 hr after hemorrhage, significant increases were observed in mRNA levels for IL-1 alpha, IL-1 beta, IL-5, and TGF-beta in intraparenchymal pulmonary mononuclear cells. The levels of TGF-beta transcripts among alveolar macrophages were increased 1 hr following blood loss, and increase in IL-1 alpha transcripts was found starting 2 hr posthemorrhage. Cells from Peyer's patches showed significant increases in mRNA levels for IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TGF-beta during the 4 hr following hemorrhage. Significant increases in mRNA levels for IL-1 beta, TNF-alpha, and TGF-beta were present within 4 hr of blood loss among cells isolated from mesenteric lymph nodes. The expression of mRNA for most cytokines was not significantly altered in splenocytes or peripheral blood mononuclear cells at any time point following hemorrhage. These experiments demonstrate that blood loss, even if resuscitated, produces significant increases in proinflammatory and immunoregulatory cytokine gene transcription as early as 1 hr following hemorrhage. These posthemorrhage alterations in cytokine mRNA expression were particularly prominent at mucosal sites, suggesting a mechanism for the increased incidence of pulmonary and intestinal involvement in organ system failure following severe blood loss and injury.

  11. Tissue-, sex- and development-specific transcription profiles of eight UDP-glucuronosyltransferase genes in zebrafish (Danio rerio) and their regulation by activator of aryl hydrocarbon receptor.

    PubMed

    Christen, Verena; Fent, Karl

    2014-05-01

    UDP-Glucuronosyltransferases (Ugts) are phase II biotransformation enzymes that glucuronidate numerous endogenous and xenobiotic substrates. Based on the reported zebrafish Ugt gene repertoire, primers for the Ugt1a and Ugt1b family and for individual Ugt5a1, Ugt5a3, Ugt5a4, Ugt5a5, Ugt5c2 and Ugt5c3 were designed and applied in RT-qPCR analyses. Transcriptional expression profiles of these Ugts were analyzed in intestine, liver, gonad and brain of female and male adult zebrafish and at different embryonic developmental stages. We found tissue-, sex- and developmental-specific expression patterns for all isoforms. Throughout all tissues, the most abundant Ugts were Ugt1a, Ugt1b, Ugt5a1 and Ugt5a3. Expression during embryonic development was assessed between 24 and 120 hpf. Ugts showed a development-dependent expression. The pattern of Ugt1a, Ugt1b, Ugt5a1, Ugt5a3 and Ugt5a4 were similar with highest expression at 24 hpf followed by a decrease and rebound increase up to 120 hpf. To analyze for transcriptional regulation of Ugts by the arylhydrocarbon receptor (ahr2), zebrafish eleuthero-embryos were exposed to 5, 25 and 50μg/L benzo(a)pyrene (BaP), a model ahr2 regulator for cyp1a. Besides transcriptional induction of ahr2 and cyp1a, BaP produced a significant induction of Ugt1a, Ugt5a1, Ugt5a3 and Ugt5a5 as well as a down-regulation of Ugt1b. These data demonstrate the link between ahr2 signalling and transcriptional expression of Ugt genes. This is the first study showing transcriptional expression of eight different Ugts in tissues and during embryonic development and offers new perspectives on the involvement of Ugts in fish xenobiotic metabolism.

  12. Cloning, molecular characterization, and expression analysis of the signal transducer and activator of transcription 3 (STAT₃) gene from grass carp (Ctenopharyngodon idellus).

    PubMed

    Guo, Ting; Leng, Xiang-Jun; Wu, Xiao-Feng; Li, Jia-Le; Gao, Jian-Zhong; Li, Xiao-Qin; Gan, Tian; Wei, Jing

    2013-11-01

    Signal transducer and activator of transcription 3 (STAT₃) binds to Janus kinase 2 (JAK₂) to initiate the JAK₂/STAT₃ signal transduction pathway, which plays an important role in cancer cell proliferation, immune regulation, reproduction, lipid metabolism, and other physiological processes of the organism. In this study, the cDNA sequence of the STAT₃ gene from grass carp was cloned using RACE (rapid-amplification of cDNA ends). Twelve characteristics of the STAT₃ gene and its encoded protein sequence were predicted and analyzed using bioinformatics methods; these features included the general physical and chemical properties, the hydrophobicity, the secondary structure and the three-dimensional structure of the protein. Quantitative real-time PCR was employed to detect grass carp STAT₃ expression pattern in different tissues. The results showed that the full-length STAT₃ gene from grass carp is 2739-bp long and contains a 216-bp 5'UTR, a 300-bp 3'UTR, and a 2223-bp open reading frame (ORF) that encodes a 740-amino acid peptide. The deduced protein exhibited 99%∼94% homology to the STAT₃ protein of zebrafish (Danio rerio), medaka (Oryzias latipes), turbot (Scophthalmus maximus), white-spotted char (Salvelinus leucomaenis), mandarin fish (Siniperca chuatsi), rainbow trout (Oncorhynchus mykiss), and green pufferfish (Tetraodon fluviatilis). The deduced grass carp STAT₃ protein contains a protein interaction domain, an alpha domain, a DNA binding domain, and an SH2 domain. The STAT₃ protein of grass carp is a hydrophilic and non-secretory protein, and its molecular mass and isoeletronic point were found to be 98,5412.1 Da and 6.39, respectively. The structural elements of STAT₃ included α-helixes, β-sheets, and loops. The grass carp STAT₃ is expressed in all of the six tissues tested, which were the liver, spleen, gill, muscle, heart, and brain. The highest expression level was found in the liver (P < 0.05), whereas a significantly

  13. Molecular genetic responses to lysergic acid diethylamide include transcriptional activation of MAP kinase phosphatase-1, C/EBP-beta and ILAD-1, a novel gene with homology to arrestins.

    PubMed

    Nichols, Charles D; Sanders-Bush, Elaine

    2004-08-01

    We recently demonstrated that the potent hallucinogenic drug lysergic acid diethylamide (LSD) dynamically influences the expression of a small collection of genes within the mammalian prefrontal cortex. Towards generating a greater understanding of the molecular genetic effects of hallucinogens and how they may relate to alterations in behavior, we have identified and characterized expression patterns of a new collection of three genes increased in expression by acute LSD administration. These genes were identified through additional screens of Affymetrix DNA microarrays and examined in experiments to assess dose-response, time course and the receptor mediating the expression changes. The first induced gene, C/EBP-beta, is a transcription factor. The second gene, MKP-1, suggests that LSD activates the MAP (mitogen activated protein) kinase pathway. The third gene, ILAD-1, demonstrates sequence similarity to the arrestins. The increase in expression of each gene was partially mediated through LSD interactions at 5-HT2A (serotonin) receptors. There is evidence of alternative splicing at the ILAD-1 locus. Furthermore, data suggests that various splice isoforms of ILAD-1 respond differently at the transcriptional level to LSD. The genes thus far found to be responsive to LSD are beginning to give a more complete picture of the complex intracellular events initiated by hallucinogens.

  14. Maternal transfer and transcriptional onset of immune genes during ontogenesis in Atlantic cod.

    PubMed

    Seppola, Marit; Johnsen, Hanne; Mennen, Saskia; Myrnes, Bjørnar; Tveiten, Helge

    2009-11-01

    The immune system in teleosts is not completely developed during embryonic and larval stages and immune competence is assumed to be restricted. This study is the first to address whether immune transcripts are maternally transferred to offspring and when immune genes are transcriptionally active in Atlantic cod (Gadus morhua). In unfertilised eggs, transcripts encoding lysozyme and cathelicidin were found indicating maternal transfer of antibacterial transcripts. Lysozyme activity was also present at this stage suggesting the presence of a functional protein. Transcripts of two other putative antibacterial genes (hepcidin and pentraxin) and antiviral genes (ISG15 and LGP2) were absent in unfertilised eggs. The transcriptional onset of these genes occurred during the gastrula period. Transcripts of the heavy chain constant regions of the immunoglobulin (Ig) D, membrane-associated and secreted form of IgM were absent in unfertilised eggs. Transcription of the heavy chain locus commenced at low levels during the segmentation period indicating the onset of B-cell development. Most innate immune genes showed an increase in transcription around hatch and first feeding, indicating a preparation for increased pathogen exposure at this time. Prior to and during metamorphosis all genes showed a pronounced elevation in transcript levels indicating a further maturation of the immune system during this period.

  15. Transcriptional activation of the SH2D2A gene is dependent on a cyclic adenosine 5'-monophosphate-responsive element in the proximal SH2D2A promoter.

    PubMed

    Dai, Ke-Zheng; Johansen, Finn-Eirik; Kolltveit, Kristin Melkevik; Aasheim, Hans-Christian; Dembic, Zlatko; Vartdal, Frode; Spurkland, Anne

    2004-05-15

    The SH2D2A gene, encoding the T cell-specific adapter protein (TSAd), is rapidly induced in activated T cells. In this study we investigate the regulation of the SH2D2A gene in Jurkat T cells and in primary T cells. Reporter gene assays demonstrated that the proximal 1-kb SH2D2A promoter was constitutively active in Jurkat TAg T cells and, to a lesser extent, in K562 myeloid cells, Reh B cells, and 293T fibroblast cells. The minimal SH2D2A promoter was located between position -236 and -93 bp from the first coding ATG, and transcriptional activity in primary T cells depended on a cAMP response element (CRE) centered around position -117. Nuclear extracts from Jurkat TAg cells and activated primary T cells contained binding activity to this CRE, as observed in an EMSA. Consistent with this observation, we found that a cAMP analog was a very potent inducer of SH2D2A mRNA expression in primary T cells as measured by real-time RT-PCR. Furthermore, activation of SH2D2A expression by CD3 stimulation required cAMP-dependent protein kinase activity. Thus, transcriptional regulation of the SH2D2A gene in activated T cells is critically dependent on a CRE in the proximal promoter region.

  16. Correcting Transcription Factor Gene Sets for Copy Number and Promoter Methylation Variations

    PubMed Central

    Rathi, Komal S.; Gaykalova, Daria A.; Hennesey, Patrick; Califano, Joseph A.; Ochs, Michael F.

    2014-01-01

    Gene set analysis provides a method to generate statistical inferences across sets of linked genes, primarily using high-throughput expression data. Common gene sets include biological pathways, operons, and targets of transcriptional regulators. In higher eukaryotes, especially when dealing with diseases with strong genetic and epigenetic components such as cancer, copy number loss and gene silencing through promoter methylation can eliminate the possibility that a gene is transcribed. This, in turn, can adversely affect the estimation of transcription factor or pathway activity from a set of target genes, since some of the targets may not be responsive to transcriptional regulation. Here we introduce a simple filtering approach that removes genes from consideration if they show copy number loss or promoter methylation and demonstrate the improvement in inference of transcription factor activity in a simulated data set based on the background expression observed in normal head and neck tissue. PMID:25195578

  17. Correcting transcription factor gene sets for copy number and promoter methylation variations.

    PubMed

    Rathi, Komal S; Gaykalova, Daria A; Hennessey, Patrick; Califano, Joseph A; Ochs, Michael F

    2014-09-01

    Gene set analysis provides a method to generate statistical inferences across sets of linked genes, primarily using high-throughput expression data. Common gene sets include biological pathways, operons, and targets of transcriptional regulators. In higher eukaryotes, especially when dealing with diseases with strong genetic and epigenetic components such as cancer, copy number loss and gene silencing through promoter methylation can eliminate the possibility that a gene is transcribed. This, in turn, can adversely affect the estimation of transcription factor or pathway activity from a set of target genes, as some of the targets may not be responsive to transcriptional regulation. Here we introduce a simple filtering approach that removes genes from consideration if they show copy number loss or promoter methylation, and demonstrate the improvement in inference of transcription factor activity in a simulated dataset based on the background expression observed in normal head and neck tissue.

  18. Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak.

    PubMed

    Hummel, Aaron W; Doyle, Erin L; Bogdanove, Adam J

    2012-09-01

    Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements.

  19. Transcription factor regulation can be accurately predicted from the presence of target gene signatures in microarray gene expression data

    PubMed Central

    Essaghir, Ahmed; Toffalini, Federica; Knoops, Laurent; Kallin, Anders; van Helden, Jacques; Demoulin, Jean-Baptiste

    2010-01-01

    Deciphering transcription factor networks from microarray data remains difficult. This study presents a simple method to infer the regulation of transcription factors from microarray data based on well-characterized target genes. We generated a catalog containing transcription factors associated with 2720 target genes and 6401 experimentally validated regulations. When it was available, a distinction between transcriptional activation and inhibition was included for each regulation. Next, we built a tool (www.tfacts.org) that compares submitted gene lists with target genes in the catalog to detect regulated transcription factors. TFactS was validated with published lists of regulated genes in various models and compared to tools based on in silico promoter analysis. We next analyzed the NCI60 cancer microarray data set and showed the regulation of SOX10, MITF and JUN in melanomas. We then performed microarray experiments comparing gene expression response of human fibroblasts stimulated by different growth factors. TFactS predicted the specific activation of Signal transducer and activator of transcription factors by PDGF-BB, which was confirmed experimentally. Our results show that the expression levels of transcription factor target genes constitute a robust signature for transcription factor regulation, and can be efficiently used for microarray data mining. PMID:20215436

  20. SUMOylation-disrupting WAS mutation converts WASp from a transcriptional activator to a repressor of NF-κB response genes in T cells.

    PubMed

    Sarkar, Koustav; Sadhukhan, Sanjoy; Han, Seong-Su; Vyas, Yatin M

    2015-10-01

    In Wiskott-Aldrich syndrome (WAS), immunodeficiency and autoimmunity often comanifest, yet how WAS mutations misregulate chromatin-signaling in Thelper (TH) cells favoring development of auto-inflammation over protective immunity is unclear. Previously, we identified an essential promoter-specific, coactivator role of nuclear-WASp in TH1 gene transcription. Here we identify small ubiquitin-related modifier (SUMO)ylation as a novel posttranslational modification of WASp, impairment of which converts nuclear-WASp from a transcriptional coactivator to a corepressor of nuclear factor (NF)-κB response genes in human (TH)1-differentiating cells. V75M, one of many disease-causing mutations occurring in SUMO*motif (72-ψψψψKDxxxxSY-83) of WASp, compromises WASp-SUMOylation, associates with COMMD1 to attenuate NF-κB signaling, and recruits histone deacetylases-6 (HDAC6) to p300-marked promoters of NF-κB response genes that pattern immunity but not inflammation. Consequently, proteins mediating adaptive immunity (IFNG, STAT1, TLR1) are deficient, whereas those mediating auto-inflammation (GM-CSF, TNFAIP2, IL-1β) are paradoxically increased in TH1 cells expressing SUMOylation-deficient WASp. Moreover, SUMOylation-deficient WASp favors ectopic development of the TH17-like phenotype (↑IL17A, IL21, IL22, IL23R, RORC, and CSF2) under TH1-skewing conditions, suggesting a role for WASp in modulating TH1/TH17 plasticity. Notably, pan-histone deacetylase inhibitors lift promoter-specific repression imposed by SUMOylation-deficient WASp and restore misregulated gene expression. Our findings uncovering a SUMOylation-based mechanism controlling WASp's dichotomous roles in transcription may have implications for personalized therapy for patients carrying mutations that perturb WASp-SUMOylation.

  1. SUMOylation-disrupting WAS mutation converts WASp from a transcriptional activator to a repressor of NF-κB response genes in T cells

    PubMed Central

    Sarkar, Koustav; Sadhukhan, Sanjoy; Han, Seong-Su

    2015-01-01

    In Wiskott-Aldrich syndrome (WAS), immunodeficiency and autoimmunity often comanifest, yet how WAS mutations misregulate chromatin-signaling in Thelper (TH) cells favoring development of auto-inflammation over protective immunity is unclear. Previously, we identified an essential promoter-specific, coactivator role of nuclear-WASp in TH1 gene transcription. Here we identify small ubiquitin-related modifier (SUMO)ylation as a novel posttranslational modification of WASp, impairment of which converts nuclear-WASp from a transcriptional coactivator to a corepressor of nuclear factor (NF)-κB response genes in human (TH)1-differentiating cells. V75M, one of many disease-causing mutations occurring in SUMO*motif (72-ψψψψKDxxxxSY-83) of WASp, compromises WASp-SUMOylation, associates with COMMD1 to attenuate NF-κB signaling, and recruits histone deacetylases-6 (HDAC6) to p300-marked promoters of NF-κB response genes that pattern immunity but not inflammation. Consequently, proteins mediating adaptive immunity (IFNG, STAT1, TLR1) are deficient, whereas those mediating auto-inflammation (GM-CSF, TNFAIP2, IL-1β) are paradoxically increased in TH1 cells expressing SUMOylation-deficient WASp. Moreover, SUMOylation-deficient WASp favors ectopic development of the TH17-like phenotype (↑IL17A, IL21, IL22, IL23R, RORC, and CSF2) under TH1-skewing conditions, suggesting a role for WASp in modulating TH1/TH17 plasticity. Notably, pan-histone deacetylase inhibitors lift promoter-specific repression imposed by SUMOylation-deficient WASp and restore misregulated gene expression. Our findings uncovering a SUMOylation-based mechanism controlling WASp’s dichotomous roles in transcription may have implications for personalized therapy for patients carrying mutations that perturb WASp-SUMOylation. PMID:26261240

  2. Gene expression in plant mitochondria: transcriptional and post-transcriptional control.

    PubMed Central

    Binder, Stefan; Brennicke, Axel

    2003-01-01

    The informational content of the mitochondrial genome in plants is, although small, essential for each cell. Gene expression in these organelles involves a number of distinct transcriptional and post-transcriptional steps. The complex post-transcriptional processes of plant mitochondria such as 5' and 3' RNA processing, intron splicing, RNA editing and controlled RNA stability extensively modify individual steady-state RNA levels and influence the mRNA quantities available for translation. In this overview of the processes in mitochondrial gene expression, we focus on confirmed and potential sites of regulatory interference and discuss the evolutionary origins of the transcriptional and post-transcriptional processes. PMID:12594926

  3. The effect of specific rhlA-las-box mutations on DNA binding and gene activation by Pseudomonas aeruginosa quorum-sensing transcriptional regulators RhlR and LasR.

    PubMed

    González-Valdez, Abigail; Servín-González, Luis; Juárez, Katy; Hernandez-Aligio, Alberto; Soberón-Chávez, Gloria

    2014-07-01

    Pseudomonas aeruginosa is a free-living bacterium and an important opportunistic pathogen. The genes coding for virulence-associated traits are regulated at the level of transcription by the quorum-sensing response. In this response, the regulator LasR coupled with the autoinducer 3-oxo-dodecanoyl homoserine lactone (3O-C12-HSL) activates transcription of genes for several virulence factors. LasR/3O-C12-HSL also activates transcription of rhlR, the gene coding for the transcriptional regulator RhlR, and of rhlI that encodes the synthase that produces the autoinducer butanoyl-homoserine lactone (C4-HSL) that interacts with RhlR. Genes activated by RhlR/C4-HSL include those involved in rhamnolipids production (like the rhlAB operon) and lecA, coding for PA-I lectin. The molecular basis of LasR/3O-C12-HSL- and RhlR/C4-HSLDNA-binding specificity (at the so-called las-boxes) has not been clearly determined, and the aim of this work was to contribute to its understanding. Therefore, we analyzed the interaction of LasR and RhlR to variants of the rhlA-las-box that were constructed based on the comparison of this las-box to the las-box of lecA. We conclude that LasR and RhlR DNA-binding specificity is a complex multifactorial phenomenon in which both positive and negative effects are involved and that binding of these proteins does not necessarily result in gene activation.

  4. Anticancer activity of Phyllanthus emblica Linn. (Indian gooseberry): inhibition of transcription factor AP-1 and HPV gene expression in cervical cancer cells.

    PubMed

    Mahata, Sutapa; Pandey, Arvind; Shukla, Shirish; Tyagi, Abhishek; Husain, Syed Akhtar; Das, Bhudev Chandra; Bharti, Alok Chandra

    2013-01-01

    Plant products of Phyllanthus emblica Linn. are traditionally consumed for its immense nutritive and medicinal values. However, the molecular mechanism(s) by which it exerts it effects is less understood. In this study, we investigated mechanism of action of P. emblica fruit extract (PE) by studying its effect on activator protein-1 (AP-1) activity and human papillomavirus (HPV) transcription that are essential for tumorigenicity of cervical cancer cells. PE resulted in a dose-and time-dependent inhibition of DNA binding activity of constitutively active AP-1 in both HPV16-positive (SiHa) and HPV18-positive (HeLa) cervical cancer cells. PE-induced AP-1 inhibition was found mediated through downregulation of constituent AP-1 proteins, c-Jun, JunB, JunD, and c-Fos; however, the kinetics of their inhibition varied in both the cell types. Inhibition of AP-1 by PE was accompanied by suppression of viral transcription that resulted in growth inhibition of cervical cancer cells. Growth inhibitory activity of PE was primarily manifested through induction of apoptotic cell death. These results suggest that P. emblica exhibits its anticancer activities through inhibition of AP-1 and targets transcription of viral oncogenes responsible for development and progression of cervical cancer thus indicating its possible utility for treatment of HPV-induced cervical cancers.

  5. The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene.

    PubMed Central

    Kuzhandaivelu, N; Jones, W K; Martin, A K; Dickson, R C

    1992-01-01

    Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription. Images PMID:1569929

  6. Downregulation of E-cadherin is an essential event in activating beta-catenin/Tcf-dependent transcription and expression of its target genes in Pdcd4 knockdown cells.

    PubMed

    Wang, Q; Sun, Z-X; Allgayer, H; Yang, H-S

    2010-01-07

    We reported earlier that knockdown of tumor suppressor Pdcd4 (programed cell death 4) downregulates E-cadherin expression and activates beta-catenin/Tcf (T-cell factor)-dependent transcription in colon tumor cells. However, the underlying mechanism of these observations remains unknown. In this study, we showed that knockdown of Pdcd4 downregulates E-cadherin expression through elevated protein level of Snail. Over-expression of Pdcd4 upregulates E-cadherin expression and inhibits beta-catenin/Tcf-dependent transcription. We then showed that knockdown of E-cadherin activates beta-catenin/Tcf-dependent transcription. Conversely, over-expression of E-cadherin in Pdcd4 knockdown cells inhibits beta-catenin/Tcf-dependent transcription. In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells. Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells. Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown. Taken together, our data showed that elevated Snail expression by Pdcd4 knockdown leads to downregulation of E-cadherin resulting in activating beta-catenin/Tcf-dependent transcription and stimulating the expression of c-Myc and u-PAR, thus providing molecular explanation of how Pdcd4 suppresses tumor invasion.

  7. Early-late genes of the ecdysone cascade as models for transcriptional studies

    PubMed Central

    Mazina, Marina Yu; Nikolenko, Julia V; Fursova, Nadezda A; Nedil'ko, Petr N; Krasnov, Aleksey N; Vorobyeva, Nadezhda E

    2015-01-01

    The DHR3 and Hr4 early-late genes of the ecdysone cascade are described as models for transcriptional studies in Drosophila cells. In a set of experiments, it became clear that these genes are a convenient and versatile system for research into the physiological conditions upon 20-hydroxyecdysone induction. DHR3 and Hr4 gene transcription is characterized by fast activation kinetics, which enables transcriptional studies without the influence of indirect effects. A limited number of activated genes (only 73 genes are induced one hour after treatment) promote the selectivity of transcriptional studies via 20-hydroxyecdysone induction. DHR3 and Hr4 gene expression is dose dependent, is completely controlled by the hormone titer and decreases within hours of 20-hydroxyecdysone withdrawal. The DHR3 and Hr4 gene promoters become functional within 20 minutes after induction, which makes them useful tools for investigation if the early activation process. Their transcription is controlled by the RNA polymerase II pausing mechanism, which is widespread in the genome of Drosophila melanogaster but is still underinvestigated. Uniform expression activation of the DHR3 and Hr4 genes in a cell population was confirmed at both the RNA and protein levels. Homogeneity of the transcription response makes DHR3/Hr4 system valuable for investigation of the protein dynamics during transcription induction. PMID:26506480

  8. Transcription of antifreeze protein genes in Choristoneura fumiferana.

    PubMed

    Qin, W; Doucet, D; Tysh