Science.gov

Sample records for silencing transcriptional bias

  1. 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

  2. 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.

  3. 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

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

    PubMed Central

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

    2016-01-01

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

  5. Traffic into silence: endomembranes and post-transcriptional RNA silencing

    PubMed Central

    Kim, Yun Ju; Maizel, Alexis; Chen, Xuemei

    2014-01-01

    microRNAs (miRNAs) and small interfering RNAs (siRNAs) are small RNAs that repress gene expression at the post-transcriptional level in plants and animals. Small RNAs guide Argonaute-containing RNA-induced silencing complexes to target RNAs in a sequence-specific manner, resulting in mRNA deadenylation followed by exonucleolytic decay, mRNA endonucleolytic cleavage, or translational inhibition. Although our knowledge of small RNA biogenesis, turnover, and mechanisms of action has dramatically expanded in the past decade, the subcellular location of small RNA-mediated RNA silencing still needs to be defined. In contrast to the prevalent presumption that RNA silencing occurs in the cytosol, emerging evidence reveals connections between the endomembrane system and small RNA activities in plants and animals. Here, we summarize the work that uncovered this link between small RNAs and endomembrane compartments and present an overview of the involvement of the endomembrane system in various aspects of RNA silencing. We propose that the endomembrane system is an integral component of RNA silencing that has been long overlooked and predict that a marriage between cell biology and RNA biology holds the key to a full understanding of post-transcriptional gene regulation by small RNAs. PMID:24668229

  6. Transgenerational analysis of transcriptional silencing in zebrafish

    PubMed Central

    Akitake, Courtney M.; Macurak, Michelle; Halpern, Marnie E.; Goll, Mary G.

    2011-01-01

    The yeast Gal4/UAS transcriptional activation system is a powerful tool for regulating gene expression in Drosophila and has been increasing in popularity for developmental studies in zebrafish. It is also useful for studying the basis of de novo transcriptional silencing. Fluorescent reporter genes under the control of multiple tandem copies of the upstream activator sequence (UAS) often show evidence of variegated expression and DNA methylation in transgenic zebrafish embryos. To characterize this systematically, we monitored the progression of transcriptional silencing of UAS-regulated transgenes that differ in their integration sites and in the repetitive nature of the UAS. Transgenic larvae were examined in three generations for tissue-specific expression of a green fluorescent protein (GFP) reporter and DNA methylation at the UAS. Single insertions containing four distinct upstream activator sequences were far less susceptible to methylation than insertions containing fourteen copies of the same UAS. In addition, transgenes that integrated in or adjacent to transposon sequence exhibited silencing regardless of the number of UAS sites included in the transgene. Placement of promoter-driven Gal4 upstream of UAS-regulated responder genes in a single bicistronic construct also appeared to accelerate silencing and methylation. The results demonstrate the utility of the zebrafish for efficient tracking of gene silencing mechanisms across several generations, as well as provide useful guidelines for optimal Gal4-regulated gene expression in organisms subject to DNA methylation. PMID:21223961

  7. Transcriptional Silencing by Polycomb-Group Proteins

    PubMed Central

    Grossniklaus, Ueli; Paro, Renato

    2014-01-01

    Polycomb-group (PcG) genes encode chromatin proteins involved in stable and heritable transcriptional silencing. PcG proteins participate in distinct multimeric complexes that deposit, or bind to, specific histone modifications (e.g., H3K27me3 and H2AK119ub1) to prevent gene activation and maintain repressed chromatin domains. PcG proteins are evolutionary conserved and play a role in processes ranging from vernalization and seed development in plants, over X-chromosome inactivation in mammals, to the maintenance of stem cell identity. PcG silencing is medically relevant as it is often observed in human disorders, including cancer, and tissue regeneration, which involve the reprogramming of PcG-controlled target genes. PMID:25367972

  8. Integrated Circuits: How Transcriptional Silencing and Counter-Silencing Facilitate Bacterial Evolution

    PubMed Central

    Will, W. Ryan; Navarre, William W.; Fang, Ferric C.

    2014-01-01

    Horizontal gene transfer is a major contributor to bacterial evolution and diversity. For a bacterial cell to utilize newly-acquired traits such as virulence and antibiotic resistance, new genes must be integrated into the existing regulatory circuitry to allow appropriate expression. Xenogeneic silencing of horizontally-acquired genes by H-NS or other nucleoid-associated proteins avoids adventitious expression and can be relieved by other DNA-binding counter-silencing proteins in an environmentally- and physiologically-responsive manner. Biochemical and genetic analyses have recently demonstrated that counter-silencing can occur at a variety of promoter architectures, in contrast to classical transcriptional activation. Disruption of H-NS nucleoprotein filaments by DNA bending is a suggested mechanism by which silencing can be relieved. This review discusses recent advances in our understanding of the mechanisms and importance of xenogeneic silencing and counter-silencing in the successful integration of horizontally-acquired genes into regulatory networks. PMID:25461567

  9. Intergenic transcription through a polycomb group response element counteracts silencing.

    PubMed

    Schmitt, Sabine; Prestel, Matthias; Paro, Renato

    2005-03-15

    Polycomb group response elements (PREs) mediate the mitotic inheritance of gene expression programs and thus maintain determined cell fates. By default, PREs silence associated genes via the targeting of Polycomb group (PcG) complexes. Upon an activating signal, however, PREs recruit counteracting trithorax group (trxG) proteins, which in turn maintain target genes in a transcriptionally active state. Using a transgenic reporter system, we show that the switch from the silenced to the activated state of a PRE requires noncoding transcription. Continuous transcription through the PRE induced by an actin promoter prevents the establishment of PcG-mediated silencing. The maintenance of epigenetic activation requires transcription through the PRE to proceed at least until embryogenesis is completed. At the homeotic bithorax complex of Drosophila, intergenic PRE transcripts can be detected not only during embryogenesis, but also at late larval stages, suggesting that transcription through endogenous PREs is required continuously as an anti-silencing mechanism to prevent the access of repressive PcG complexes to the chromatin. Furthermore, all other PREs outside the homeotic complex we tested were found to be transcribed in the same tissue as the mRNA of the corresponding target gene, suggesting that anti-silencing by transcription is a fundamental aspect of the cellular memory system.

  10. Post-transcriptional gene silencing activity of human GIGYF2.

    PubMed

    Kryszke, Marie-Hélène; Adjeriou, Badia; Liang, Feifei; Chen, Hong; Dautry, François

    2016-07-01

    In mammalian post-transcriptional gene silencing, the Argonaute protein AGO2 indirectly recruits translation inhibitors, deadenylase complexes, and decapping factors to microRNA-targeted mRNAs, thereby repressing mRNA translation and accelerating mRNA decay. However, the exact composition and assembly pathway of the microRNA-induced silencing complex are not completely elucidated. As the GYF domain of human GIGYF2 was shown to bind AGO2 in pulldown experiments, we wondered whether GIGYF2 could be a novel protein component of the microRNA-induced silencing complex. Here we show that full-length GIGYF2 coimmunoprecipitates with AGO2 in human cells, and demonstrate that, upon tethering to a reporter mRNA, GIGYF2 exhibits strong, dose-dependent silencing activity, involving both mRNA destabilization and translational repression. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Endogenous Targets of Transcriptional Gene Silencing in Arabidopsis

    PubMed Central

    Steimer, Andrea; Amedeo, Paolo; Afsar, Karin; Fransz, Paul; Scheid, Ortrun Mittelsten; Paszkowski, Jerzy

    2000-01-01

    Transcriptional gene silencing (TGS) frequently inactivates foreign genes integrated into plant genomes but very likely also suppresses an unknown subset of chromosomal information. Accordingly, RNA analysis of mutants impaired in silencing should uncover endogenous targets of this epigenetic regulation. We compared transcripts from wild-type Arabidopsis carrying a silent transgene with RNA from an isogenic transgene-expressing TGS mutant. Two cDNA clones were identified representing endogenous RNA expressed only in the mutant. The synthesis of these RNAs was found to be released in several mutants affected in TGS, implying that TGS in general and not a particular mutation controls the transcriptional activity of their templates. Detailed analysis revealed that the two clones are part of longer transcripts termed TSI (for transcriptionally silent information). Two major classes of related TSI transcripts were found in a mutant cDNA library. They are synthesized from repeats present in heterochromatic pericentromeric regions of Arabidopsis chromosomes. These repeats share sequence homology with the 3′ terminal part of the putative retrotransposon Athila. However, the transcriptional activation does not include the transposon itself and does not promote its movement. There is no evidence for a general release of silencing from retroelements. Thus, foreign genes in plants encounter the epigenetic control normally directed, at least in part, toward a subset of pericentromeric repeats. PMID:10899982

  12. Rhodopsin targeted transcriptional silencing by DNA-binding.

    PubMed

    Botta, Salvatore; Marrocco, Elena; de Prisco, Nicola; Curion, Fabiola; Renda, Mario; Sofia, Martina; Lupo, Mariangela; Carissimo, Annamaria; Bacci, Maria Laura; Gesualdo, Carlo; Rossi, Settimio; Simonelli, Francesca; Surace, Enrico Maria

    2016-03-14

    Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations.

  13. Rhodopsin targeted transcriptional silencing by DNA-binding

    PubMed Central

    Botta, Salvatore; Marrocco, Elena; de Prisco, Nicola; Curion, Fabiola; Renda, Mario; Sofia, Martina; Lupo, Mariangela; Carissimo, Annamaria; Bacci, Maria Laura; Gesualdo, Carlo; Rossi, Settimio; Simonelli, Francesca; Surace, Enrico Maria

    2016-01-01

    Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations. DOI: http://dx.doi.org/10.7554/eLife.12242.001 PMID:26974343

  14. The RNA-induced transcriptional silencing complex targets chromatin exclusively via interacting with nascent transcripts

    PubMed Central

    Shimada, Yukiko; Mohn, Fabio; Bühler, Marc

    2016-01-01

    Small RNAs regulate chromatin modification and transcriptional gene silencing across the eukaryotic kingdom. Although these processes have been well studied, fundamental mechanistic aspects remain obscure. Specifically, it is unclear exactly how small RNA-loaded Argonaute protein complexes target chromatin to mediate silencing. Here, using fission yeast, we demonstrate that transcription of the target locus is essential for RNA-directed formation of heterochromatin. However, high transcriptional activity is inhibitory; thus, a transcriptional window exists that is optimal for silencing. We further found that pre-mRNA splicing is compatible with RNA-directed heterochromatin formation. However, the kinetics of pre-mRNA processing is critical. Introns close to the 5′ end of a transcript that are rapidly spliced result in a bistable response whereby the target either remains euchromatic or becomes fully silenced. Together, our results discount siRNA–DNA base pairing in RNA-mediated heterochromatin formation, and the mechanistic insights further reveal guiding paradigms for the design of small RNA-directed chromatin silencing studies in multicellular organisms. PMID:27941123

  15. Local infiltration of high- and low-molecular-weight RNA from silenced sunflower (Helianthus annuus L.) plants triggers post-transcriptional gene silencing in non-silenced plants.

    PubMed

    Hewezi, Tarek; Alibert, Gilbert; Kallerhoff, Jean

    2005-01-01

    Using grafting procedures, we have characterized post-transcriptional gene silencing (PTGS) in transgenic sunflower expressing beta-glucuronidase (GUS) activity. Silencing was observed as early as 2 weeks after grafting of non-silenced scions on to silenced rootstock. Transmission of the systemic signal occurs solely from stock to scion, is independent of the physiological age of the rootstock and is not heritable. Furthermore, we report, for the first time in plants, an easy and low-cost method of activating RNA silencing by infiltration of purified RNA from silenced plants. Local application of total RNA derived from silenced sunflower plants to leaves of non-silenced plants induces PTGS in newly developed leaves above the point of infiltration, as shown by reduced GUS activity and mRNA levels. Silenced plants contain 21-23-nucleotide RNAs hybridizing to transgene target sequences, in contrast with leaves of non-silenced plants. However, de novo production of GUS-specific short RNA in non-silenced plants can be activated by leaf infiltration of low-molecular-weight RNAs isolated from leaves of silenced plants. Significant levels were detected as early as 2 weeks after infiltration, peaked at 3 weeks and declined 5 weeks after infiltration. Our results provide evidence that RNA infiltration in sunflower induces transient silencing and is not transmitted to offspring. This approach could be of major use in dissecting the mechanisms involved in PTGS.

  16. Sequence homology requirements for transcriptional silencing of 35S transgenes and post-transcriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus.

    PubMed

    Thierry, D; Vaucheret, H

    1996-12-01

    The transgene locus of the tobacco plant 271 (271 locus) is located on a telomere and consists of multiple copies of a plasmid carrying an NptII marker gene driven by the cauliflower mosaic virus (CaMV) 19S promoter and the leaf-specific nitrite reductase Nii1 cDNA cloned in the antisense orientation under the control of the CaMV 35S promoter. Previous analysis of gene expression in leaves has shown that this locus triggers both post-transcriptional silencing of the host leaf-specific Nii genes and transcriptional silencing of transgenes driven by the 19S or 35S promoter irrespective of their coding sequence and of their location in the genome. In this paper we show that silencing of transgenes carrying Nii1 sequences occurs irrespective of the promoter driving their expression and of their location within the genome. This phenomenon occurs in roots as well as in leaves although root Nii genes share only 84% identity with leaf-specific Nii1 sequences carried by the 271 locus. Conversely, transgenes carrying the bean Nii gene (which shares 76% identity with the tobacco Nii1 gene) escape silencing by the 271 locus. We also show that transgenes driven by the figwort mosaic virus 34S promoter (which shares 63% identity with the 35S promoter) also escape silencing by the 271 locus. Taken together, these results indicate that a high degree of sequence similarity is required between the sequences of the silencing locus and of the target (trans)genes for both transcriptional and post-transcriptional silencing.

  17. Trans-Reactivation: A New Epigenetic Phenomenon Underlying Transcriptional Reactivation of Silenced Genes

    PubMed Central

    Cavalieri, Vincenzo; Ingrassia, Antonia M. R.; Pavesi, Giulio; Corona, Davide F. V.

    2015-01-01

    In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin. We found that non-functional alleles of white, unable to produce a coding transcript, could reactivate in trans the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional trans-reactivation is heritable, relies on the presence of homologous RNA’s and is affected by mutations in genes involved in post-transcriptional gene silencing. Our data suggest a general new unexpected level of gene expression control mediated by homologous RNA molecules in the context of heterochromatic genes. PMID:26292210

  18. A region of the nucleosome required for multiple types of transcriptional silencing in Saccharomyces cerevisiae.

    PubMed

    Prescott, Eugenia T; Safi, Alexias; Rusche, Laura N

    2011-07-01

    Extended heterochromatin domains, which are repressive to transcription and help define centromeres and telomeres, are formed through specific interactions between silencing proteins and nucleosomes. This study reveals that in Saccharomyces cerevisiae, the same nucleosomal surface is critical for the formation of multiple types of heterochromatin, but not for local repression mediated by a related transcriptional repressor. Thus, this region of the nucleosome may be generally important to long-range silencing. In S. cerevisiae, the Sir proteins perform long-range silencing, whereas the Sum1 complex acts locally to repress specific genes. A mutant form of Sum1p, Sum1-1p, achieves silencing in the absence of Sir proteins. A genetic screen identified mutations in histones H3 and H4 that disrupt Sum1-1 silencing and fall in regions of the nucleosome previously known to disrupt Sir silencing and rDNA silencing. In contrast, no mutations were identified that disrupt wild-type Sum1 repression. Mutations that disrupt silencing fall in two regions of the nucleosome, the tip of the H3 tail and a surface of the nucleosomal core (LRS domain) and the adjacent base of the H4 tail. The LRS/H4 tail region interacts with the Sir3p bromo-adjacent homology (BAH) domain to facilitate Sir silencing. By analogy, this study is consistent with the LRS/H4 tail region interacting with Orc1p, a paralog of Sir3p, to facilitate Sum1-1 silencing. Thus, the LRS/H4 tail region of the nucleosome may be relatively accessible and facilitate interactions between silencing proteins and nucleosomes to stabilize long-range silencing.

  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. Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor

    PubMed Central

    Paonessa, Francesco; Criscuolo, Stefania; Sacchetti, Silvio; Amoroso, Davide; Scarongella, Helena; Pecoraro Bisogni, Federico; Carminati, Emanuele; Pruzzo, Giacomo; Maragliano, Luca; Cesca, Fabrizia; Benfenati, Fabio

    2016-01-01

    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. PMID:26699507

  1. YY1 binding association with sex-biased transcription revealed through X-linked transcript levels and allelic binding analyses

    PubMed Central

    Chen, Chih-yu; Shi, Wenqiang; Balaton, Bradley P.; Matthews, Allison M.; Li, Yifeng; Arenillas, David J.; Mathelier, Anthony; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R. R.; Brown, Carolyn J.; Wasserman, Wyeth W.

    2016-01-01

    Sex differences in susceptibility and progression have been reported in numerous diseases. Female cells have two copies of the X chromosome with X-chromosome inactivation imparting mono-allelic gene silencing for dosage compensation. However, a subset of genes, named escapees, escape silencing and are transcribed bi-allelically resulting in sexual dimorphism. Here we conducted in silico analyses of the sexes using human datasets to gain perspectives into such regulation. We identified transcription start sites of escapees (escTSSs) based on higher transcription levels in female cells using FANTOM5 CAGE data. Significant over-representations of YY1 transcription factor binding motif and ChIP-seq peaks around escTSSs highlighted its positive association with escapees. Furthermore, YY1 occupancy is significantly biased towards the inactive X (Xi) at long non-coding RNA loci that are frequent contacts of Xi-specific superloops. Our study suggests a role for YY1 in transcriptional activity on Xi in general through sequence-specific binding, and its involvement at superloop anchors. PMID:27857184

  2. Silencers

    NASA Astrophysics Data System (ADS)

    Kurze, U.; Riedel, E.

    Large size silencers are attached to the intake and exhaust of large industrial plants, e.g. forced ventilation systems for mining industry, intake of cooling towers (Fig. 11.1) or flue gas stacks of power plants to protect the neighbourhood from plant noise. Large silencers are also required for ventilation openings of rooms with high internal sound pressure levels, e.g. industrial production halls or subway ventilation ducts.

  3. DNA double strand break repair, chromosome synapsis and transcriptional silencing in meiosis.

    PubMed

    Inagaki, Akiko; Schoenmakers, Sam; Baarends, Willy M

    2010-05-16

    Chromosome pairing and synapsis during meiotic prophase requires the formation and repair of DNA double-strand breaks (DSBs) by the topoisomerase-like enzyme SPO11. Chromosomes, or chromosomal regions, that lack a pairing partner, such as the largely heterologous X and Y chromosomes, show delayed meiotic DSB repair and are transcriptionally silenced. Herein, we review meiosis-specific aspects of DSB repair in relation to homology recognition and meiotic silencing of heterologous regions. We propose a dynamic interplay between progression of synapsis and persistent meiotic DSBs. Signaling from these persistent breaks could inhibit heterologous synapsis and stimulate meiotic silencing of the X and Y chromosomes.

  4. Checks and balances between cohesin and polycomb in gene silencing and transcription.

    PubMed

    Dorsett, Dale; Kassis, Judith A

    2014-06-02

    The cohesin protein complex was discovered for its roles in sister chromatid cohesion and segregation, and the Polycomb group (PcG) proteins for their roles in epigenetic gene silencing during development. Cohesin also controls gene transcription via multiple mechanisms. Genetic and molecular evidence from Drosophila argue that cohesin and the PRC1 PcG complex interact to control transcription of many active genes that are critical for development, and that via these interactions cohesin also controls the availability of PRC1 for gene silencing.

  5. Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

    PubMed Central

    Pratt-Hyatt, Matthew; Pai, Dave A.; Haeusler, Rebecca A.; Wozniak, Glenn G.; Good, Paul D.; Miller, Erin L.; McLeod, Ian X.; Yates, John R.; Hopper, Anita K.; Engelke, David R.

    2013-01-01

    The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification. PMID:23898186

  6. Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing.

    PubMed

    Pratt-Hyatt, Matthew; Pai, Dave A; Haeusler, Rebecca A; Wozniak, Glenn G; Good, Paul D; Miller, Erin L; McLeod, Ian X; Yates, John R; Hopper, Anita K; Engelke, David R

    2013-08-13

    The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification.

  7. Kcnq1ot1/Lit1 Noncoding RNA Mediates Transcriptional Silencing by Targeting to the Perinucleolar Region ▿ †

    PubMed Central

    Mohammad, Faizaan; Pandey, Radha Raman; Nagano, Takashi; Chakalova, Lyubomira; Mondal, Tanmoy; Fraser, Peter; Kanduri, Chandrasekhar

    2008-01-01

    The Kcnq1ot1 antisense noncoding RNA has been implicated in long-range bidirectional silencing, but the underlying mechanisms remain enigmatic. Here we characterize a domain at the 5′ end of the Kcnq1ot1 RNA that carries out transcriptional silencing of linked genes using an episomal vector system. The bidirectional silencing property of Kcnq1ot1 maps to a highly conserved repeat motif within the silencing domain, which directs transcriptional silencing by interaction with chromatin, resulting in histone H3 lysine 9 trimethylation. Intriguingly, the silencing domain is also required to target the episomal vector to the perinucleolar compartment during mid-S phase. Collectively, our data unfold a novel mechanism by which an antisense RNA mediates transcriptional gene silencing of chromosomal domains by targeting them to distinct nuclear compartments known to be rich in heterochromatic machinery. PMID:18299392

  8. Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice.

    PubMed

    Kumpatla, S P; Teng, W; Buchholz, W G; Hall, T C

    1997-10-01

    Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btt cryIIIA and bar transgenes in rice (Oryza sativa L.) that displayed aberrant segregation in R1 progeny. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R1 plants had a DNA hybridization profile identical to that of the resistant R0 parent and R1 siblings. Genomic DNA analysis revealed substantial methylation of the Ubi1/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R2 progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubi1 promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences.

  9. V2 from a curtovirus is a suppressor of post-transcriptional gene silencing.

    PubMed

    Luna, Ana P; Rodríguez-Negrete, Edgar A; Morilla, Gabriel; Wang, Liping; Lozano-Durán, Rosa; Castillo, Araceli G; Bejarano, Eduardo R

    2017-10-01

    The suppression of gene silencing is a key mechanism for the success of viral infection in plants. DNA viruses from the Geminiviridae family encode several proteins that suppress transcriptional and post-transcriptional gene silencing (TGS/PTGS). In Begomovirus, the most abundant genus of this family, three out of six genome-encoded proteins, namely C2, C4 and V2, have been shown to suppress PTGS, with V2 being the strongest PTGS suppressor in transient assays. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among geminiviruses. In this genus, only one protein, C2/L2, has been described as inhibiting PTGS. We show here that, despite the lack of sequence homology with its begomoviral counterpart, BCTV V2 acts as a potent PTGS suppressor, possibly by impairing the RDR6 (RNA-dependent RNA polymerase 6)/suppressor of gene silencing 3 (SGS3) pathway.

  10. MicroRNAs distinguish translational from transcriptional silencing during endotoxin tolerance.

    PubMed

    El Gazzar, Mohamed; McCall, Charles E

    2010-07-02

    We reported that gene-selective formation of facultative heterochromatin silences transcription of acute inflammatory genes during endotoxin (LPS) tolerance, according to function. We discovered that reversal of the epigenetically silenced transcription restored mRNA levels but not protein synthesis. Here, we find that translation repression of tumor necrosis factor-alpha (TNFalpha) occurs independent of transcription silencing during LPS tolerance. The process required to disrupt protein synthesis followed Toll-like receptor 4 (TLR4)-dependent induction of microRNA (miR)-221, miR-579, and miR-125b, which coupled with RNA-binding proteins TTP, AUF1, and TIAR at the 3'-untranslated region to arrest protein synthesis. TTP and AUF1 proteins linked to miR-221, whereas TIAR coupled with miR-579 and miR-125b. Functional inhibition of miR-221 prevented TNFalpha mRNA degradation, and blocking miR-579 and miR-125b precluded translation arrest. The functional specificity of the TNFalpha 3'-untranslated region was demonstrated using luciferase reporter with mutations in the three putative miRNA binding sites. Post-transcriptional silencing was gene-specific, because it did not affect production of the IkappaBalpha anti-inflammatory protein. These results suggest that TLR4-dependent reprogramming of inflammatory genes is regulated at two separate and distinct levels. The first level of control is mediated by epigenetic modifications at the promoters that control transcription. The second and previously unrecognized level of control is mediated by TLR4-dependent differential expression of miRNAs that exert post-transcriptional controls. The concept of distinct regulation of transcription and translation was confirmed in murine sepsis. We conclude that transcription- and translation-repressive events combine to tightly regulate pro-inflammatory genes during LPS tolerance, a common feature of severe systemic inflammation.

  11. The splicing machinery promotes RNA-directed DNA methylation and transcriptional silencing in Arabidopsis

    PubMed Central

    Zhang, Cui-Jun; Zhou, Jin-Xing; Liu, Jun; Ma, Ze-Yang; Zhang, Su-Wei; Dou, Kun; Huang, Huan-Wei; Cai, Tao; Liu, Renyi; Zhu, Jian-Kang; He, Xin-Jian

    2013-01-01

    DNA methylation in transposons and other DNA repeats is conserved in plants as well as in animals. In Arabidopsis thaliana, an RNA-directed DNA methylation (RdDM) pathway directs de novo DNA methylation. We performed a forward genetic screen for suppressors of the DNA demethylase mutant ros1 and identified a novel Zinc-finger and OCRE domain-containing Protein 1 (ZOP1) that promotes Pol IV-dependent siRNA accumulation, DNA methylation, and transcriptional silencing. Whole-genome methods disclosed the genome-wide effects of zop1 on Pol IV-dependent siRNA accumulation and DNA methylation, suggesting that ZOP1 has both RdDM-dependent and -independent roles in transcriptional silencing. We demonstrated that ZOP1 is a pre-mRNA splicing factor that associates with several typical components of the splicing machinery as well as with Pol II. Immunofluorescence assay revealed that ZOP1 overlaps with Cajal body and is partially colocalized with NRPE1 and DRM2. Moreover, we found that the other development-defective splicing mutants tested including mac3a3b, mos4, mos12 and mos14 show defects in RdDM and transcriptional silencing. We propose that the splicing machinery rather than specific splicing factors is involved in promoting RdDM and transcriptional silencing. PMID:23524848

  12. Transcriptionally Silenced Transgenes in Maize Are Activated by Three Mutations Defective in Paramutation

    PubMed Central

    McGinnis, Karen M.; Springer, Catherine; Lin, Yan; Carey, Charles C.; Chandler, Vicki

    2006-01-01

    Plants with mutations in one of three maize genes, mop1, rmr1, and rmr2, are defective in paramutation, an allele-specific interaction that leads to meiotically heritable chromatin changes. Experiments reported here demonstrate that these genes are required to maintain the transcriptional silencing of two different transgenes, suggesting that paramutation and transcriptional silencing of transgenes share mechanisms. We hypothesize that the transgenes are silenced through an RNA-directed chromatin mechanism, because mop1 encodes an RNA-dependent RNA polymerase. In all the mutants, DNA methylation was reduced in the active transgenes relative to the silent transgenes at all of the CNG sites monitored within the transgene promoter. However, asymmetrical methylation persisted at one site within the reactivated transgene in the rmr1-1 mutant. With that one mutant, rmr1-1, the transgene was efficiently resilenced upon outcrossing to reintroduce the wild-type protein. In contrast, with the mop1-1 and rmr2-1 mutants, the transgene remained active in a subset of progeny even after the wild-type proteins were reintroduced by outcrossing. Interestingly, this immunity to silencing increased as the generations progressed, consistent with a heritable chromatin state being formed at the transgene in plants carrying the mop1-1 and rmr2-1 mutations that becomes more resistant to silencing in subsequent generations. PMID:16702420

  13. Epigenetic inheritance of transcriptional silencing and switching competence in fission yeast.

    PubMed

    Thon, G; Friis, T

    1997-03-01

    Epigenetic events allow the inheritance of phenotypic changes that are not caused by an alteration in DNA sequence. Here we characterize an epigenetic phenomenon occurring in the mating-type region of fission yeast. Cells of fission yeast switch between the P and M mating-type by interconverting their expressed mating-type cassette between two allelic forms, mat1-P and mat1-M. The switch results from gene conversions of mat1 by two silent cassettes, mat2-P and mat3-M, which are linked to each other and to mat1. GREWAL and KLAR observed that the ability to both switch mat1 and repress transcription near mat2-P and mat3-M was maintained epigenetically in a strain with an 8-kb deletion between mat2 and mat3. Using a strain very similar to theirs, we determined that interconversions between the switching- and silencing-proficient state and the switching and silencing-deficient state occurred less frequently than once per 1000 cell divisions. Although transcriptional silencing was alleviated by the 8-kb deletion, it was not abolished. We performed a mutant search and obtained a class of trans-acting mutations that displayed a strong cumulative effect with the 8-kb deletion. These mutations allow to assess the extent to which silencing is affected by the deletion and provide new insights on the redundancy of the silencing mechanism.

  14. Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing

    PubMed Central

    Zheng, Xianwu; Zhu, Jianhua; Kapoor, Avnish; Zhu, Jian-Kang

    2007-01-01

    Argonautes (AGOs) are conserved proteins that contain an RNA-binding PAZ domain and an RNase H-like PIWI domain. In Arabidopsis, except for AGO1, AGO4 and AGO7, the roles of seven other AGOs in gene silencing are not known. We found that a mutation in AGO6 partially suppresses transcriptional gene silencing in the DNA demethylase mutant ros1-1. In ago6-1ros1-1 plants, RD29A promoter short interfering RNAs (siRNAs) are less abundant, and cytosine methylation at both transgenic and endogenous RD29A promoters is reduced, compared to that in ros1-1. Interestingly, the ago4-1 mutation has a stronger suppression of the transcriptional silencing phenotype of ros1-1 mutant. Analysis of cytosine methylation at the endogenous MEA-ISR, AtREP2 and SIMPLEHAT2 loci revealed that the CpNpG and asymmetric methylation levels are lower in either of the ago6-1 and ago4-1 single mutants than those in the wild type, and the levels are the lowest in the ago6-1ago4-1 double mutant. These results suggest that AGO6 is important for the accumulation of specific heterochromatin-related siRNAs, and for DNA methylation and transcriptional gene silencing, this function is partly redundant with AGO4. PMID:17332757

  15. Transcriptional gene silencing by Arabidopsis microrchidia homologues involves the formation of heteromers.

    PubMed

    Moissiard, Guillaume; Bischof, Sylvain; Husmann, Dylan; Pastor, William A; Hale, Christopher J; Yen, Linda; Stroud, Hume; Papikian, Ashot; Vashisht, Ajay A; Wohlschlegel, James A; Jacobsen, Steven E

    2014-05-20

    Epigenetic gene silencing is of central importance to maintain genome integrity and is mediated by an elaborate interplay between DNA methylation, histone posttranslational modifications, and chromatin remodeling complexes. DNA methylation and repressive histone marks usually correlate with transcriptionally silent heterochromatin, however there are exceptions to this relationship. In Arabidopsis, mutation of Morpheus Molecule 1 (MOM1) causes transcriptional derepression of heterochromatin independently of changes in DNA methylation. More recently, two Arabidopsis homologues of mouse microrchidia (MORC) genes have also been implicated in gene silencing and heterochromatin condensation without altering genome-wide DNA methylation patterns. In this study, we show that Arabidopsis microrchidia (AtMORC6) physically interacts with AtMORC1 and with its close homologue, AtMORC2, in two mutually exclusive protein complexes. RNA-sequencing analyses of high-order mutants indicate that AtMORC1 and AtMORC2 act redundantly to repress a common set of loci. We also examined genetic interactions between AtMORC6 and MOM1 pathways. Although AtMORC6 and MOM1 control the silencing of a very similar set of genomic loci, we observed synergistic transcriptional regulation in the mom1/atmorc6 double mutant, suggesting that these epigenetic regulators act mainly by different silencing mechanisms.

  16. 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.

  17. The protein kinase TOUSLED is required for maintenance of transcriptional gene silencing in Arabidopsis

    PubMed Central

    Wang, Yu; Liu, Jun; Xia, Ran; Wang, Junguo; Shen, Jie; Cao, Rui; Hong, Xuhui; Zhu, Jian-Kang; Gong, Zhizhong

    2007-01-01

    TOUSLED-like kinases (TLKs) are highly conserved in plants and animals, but direct evidence linking TLKs and transcriptional gene silencing is lacking. We isolated two new alleles of TOUSLED (TSL). Mutations of TSL in ros1 reactivate the transcriptionally silent 35S-NPTII transgene and the transcriptionally silent endogenous loci TSI (TRANSCRIPTIONAL SILENCING INFORMATION). Chromatin immunoprecipitation (ChIP) analysis shows that histone H3Lys9 dimethylation is decreased in the reactivated transgene and endogenous TSI loci in the tsl ros1 mutant. However, there is no change in DNA methylation in the affected loci. Western blot and ChIP assay suggest that TSL might not be responsible for histone H3Ser10 phosphorylation. The tsl seedlings were more sensitive to DNA damage reagent methyl methanesulphonate and UV-B light. Our results provide direct evidence for a crucial role of the TOUSLED protein kinase in the maintenance of transcriptional gene silencing in some genomic regions in a DNA-methylation-independent manner in Arabidopsis. PMID:17110953

  18. The protein kinase TOUSLED is required for maintenance of transcriptional gene silencing in Arabidopsis.

    PubMed

    Wang, Yu; Liu, Jun; Xia, Ran; Wang, Junguo; Shen, Jie; Cao, Rui; Hong, Xuhui; Zhu, Jian-Kang; Gong, Zhizhong

    2007-01-01

    TOUSLED-like kinases (TLKs) are highly conserved in plants and animals, but direct evidence linking TLKs and transcriptional gene silencing is lacking. We isolated two new alleles of TOUSLED (TSL). Mutations of TSL in ros1 reactivate the transcriptionally silent 35S-NPTII transgene and the transcriptionally silent endogenous loci TSI (TRANSCRIPTIONAL SILENCING INFORMATION). Chromatin immunoprecipitation (ChIP) analysis shows that histone H3Lys9 dimethylation is decreased in the reactivated transgene and endogenous TSI loci in the tsl ros1 mutant. However, there is no change in DNA methylation in the affected loci. Western blot and ChIP assay suggest that TSL might not be responsible for histone H3Ser10 phosphorylation. The tsl seedlings were more sensitive to DNA damage reagent methyl methanesulphonate and UV-B light. Our results provide direct evidence for a crucial role of the TOUSLED protein kinase in the maintenance of transcriptional gene silencing in some genomic regions in a DNA-methylation-independent manner in Arabidopsis.

  19. Genetic Changes to a Transcriptional Silencer Element Confers Phenotypic Diversity within and between Drosophila Species.

    PubMed

    Johnson, Winslow C; Ordway, Alison J; Watada, Masayoshi; Pruitt, Jonathan N; Williams, Thomas M; Rebeiz, Mark

    2015-06-01

    The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting "enhancer" elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony's transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer's activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits.

  20. Transcription Regulation of Sex-Biased Genes during Ontogeny in the Malaria Vector Anopheles gambiae

    PubMed Central

    Windbichler, Nikolai; Papathanos, Philippos-Aris; Nolan, Tony; Dottorini, Tania; Rizzi, Ermanno; Christophides, George K.; Crisanti, Andrea

    2011-01-01

    In Anopheles gambiae, sex-regulated genes are responsible for controlling gender dimorphism and are therefore crucial in determining the ability of female mosquitoes to transmit human malaria. The identification and functional characterization of these genes will shed light on the sexual development and maturation of mosquitoes and provide useful targets for genetic control measures aimed at reducing mosquito fertility and/or distorting the sex ratio. We conducted a genome wide transcriptional analysis of sex-regulated genes from early developmental stages through adulthood combined with functional screening of novel gonadal genes. Our results demonstrate that the male-biased genes undergo a major transcription turnover starting from larval stages to adulthood. The male biased genes at the adult stage include a significant high number of unique sequences compared to the rest of the genome. This is in contrast to female-biased genes that are much more conserved and are mainly activated during late developmental stages. The high frequency of unique sequences would indicate that male-biased genes evolve more rapidly than the rest of the genome. This finding is particularly intriguing because A. gambiae is a strictly female monogamous species suggesting that driving forces in addition to sperm competition must account for the rapid evolution of male-biased genes. We have also identified and functionally characterized a number of previously unknown A. gambiae testis- and ovary-specific genes. Two of these genes, zero population growth and a suppressor of defective silencing 3 domain of the histone deacetylase co-repressor complex, were shown to play a key role in gonad development. PMID:21738713

  1. Fission yeast mutants that alleviate transcriptional silencing in centromeric flanking repeats and disrupt chromosome segregation.

    PubMed

    Ekwall, K; Cranston, G; Allshire, R C

    1999-11-01

    In the fission yeast Schizosaccharomyces pombe genes are transcriptionally silenced when placed within centromeres, within or close to the silent mating-type loci or adjacent to telomeres. Factors required to maintain mating-type silencing also affect centromeric silencing and chromosome segregation. We isolated mutations that alleviate repression of marker genes in the inverted repeats flanking the central core of centromere I. Mutations csp1 to 13 (centromere: suppressor of position effect) defined 12 loci. Ten of the csp mutants have no effect on mat2/3 or telomere silencing. All csp mutants allow some expression of genes in the centromeric flanking repeat, but expression in the central core is undetectable. Consistent with defective centromere structure and function, chromosome loss rates are elevated in all csp mutants. Mutants csp1 to 6 are temperature-sensitive lethal and csp3 and csp6 cells are defective in mitosis at 36 degrees. csp7 to 13 display a high incidence of lagging chromosomes on late anaphase spindles. Thus, by screening for mutations that disrupt silencing in the flanking region of a fission yeast centromere a novel collection of mutants affecting centromere architecture and chromosome segregation has been isolated.

  2. E-Cadherin Is Transcriptionally Activated via Suppression of ZEB1 Transcriptional Repressor by Small RNA-Mediated Gene Silencing

    PubMed Central

    Mazda, Minami; Nishi, Kenji; Naito, Yuki; Ui-Tei, Kumiko

    2011-01-01

    RNA activation has been reported to be induced by small interfering RNAs (siRNAs) that act on the promoters of several genes containing E-cadherin. In this study, we present an alternative mechanism of E-cadherin activation in human PC-3 cells by siRNAs previously reported to possess perfect-complementary sequences to E-cadherin promoter. We found that activation of E-cadherin can be also induced via suppression of ZEB1, which is a transcriptional repressor of E-cadherin, by seed-dependent silencing mechanism of these siRNAs. The functional seed-complementary sites of the siRNAs were found in the coding region in addition to the 3′ untranslated region of ZEB1 mRNA. Promoter analyses indicated that E-boxes, which are ZEB1-binding sites, in the upstream promoter region are indispensable for E-cadherin transcription by the siRNAs. Thus, the results caution against ignoring siRNA seed-dependent silencing effects in genome-wide transcriptional regulation. In addition, members of miR-302/372/373/520 family, which have the same seed sequences with one of the siRNAs containing perfect-complementarity to E-cadherin promoter, are also found to activate E-cadherin transcription. Thus, E-cadherin could be upregulated by the suppression of ZEB1 transcriptional repressor by miRNAs in vivo. PMID:22205962

  3. Genetic Changes to a Transcriptional Silencer Element Confers Phenotypic Diversity within and between Drosophila Species

    PubMed Central

    Watada, Masayoshi; Pruitt, Jonathan N.; Williams, Thomas M.; Rebeiz, Mark

    2015-01-01

    The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting “enhancer” elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony’s transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer’s activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits. PMID:26115430

  4. 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.

  5. Repressor element-1 silencing transcription factor (REST) is present in human control and Huntington's disease neurones.

    PubMed

    Schiffer, Davide; Caldera, Valentina; Mellai, Marta; Conforti, Paola; Cattaneo, Elena; Zuccato, Chiara

    2014-12-01

    The repressor element-1 silencing transcription factor/neurone-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST/NRSF functions by recruiting other cofactors to genomic loci that contain the repressor element 1/neurone restrictive silencer element (RE1/NRSE) binding motif. In brain, demonstration of REST protein presence in neurones has remained controversial. However, RE1/NRSE containing neuronal genes are actively modulated and REST dysregulation is implicated in Huntington's disease (HD). We aimed to investigate REST distribution in autopsy brain from control and HD patients. Brain tissues from six controls and six HD cases (Vonsattel grade 3 and 4) were investigated using immunohistochemical analysis. REST was present in neurones and glial cells of the cortex, caudate nucleus, hippocampus and cerebellum. REST labelling was mainly cytoplasmic in neurones while preferential nuclear staining of REST was found in glial cells. We also found that REST and huntingtin (HTT) colocalize in human neurones. Low levels of cytoplasmic REST were detected in neurones of the HD cortex and caudate but no direct relationship between decreased neuronal REST expression and disease grade was observed. These data support the notion of REST presence in human brain neurones and glial cells and indicate the importance of developing compounds able to restore REST-regulated transcription of neuronal genes in HD. © 2014 British Neuropathological Society.

  6. RNA Silencing Induced by an Artificial Sequence That Prevents Proper Transcription Termination in Rice1[W

    PubMed Central

    Kawakatsu, Taiji; Wakasa, Yuhya; Yasuda, Hiroshi; Takaiwa, Fumio

    2012-01-01

    Posttranscriptional gene silencing (PTGS) is a sequence-specific mRNA degradation caused by small RNA, such as microRNA (miRNA) and small interfering RNA (siRNA). miRNAs are generated from MIRNA loci, whereas siRNAs originate from various sources of double-stranded RNA. In this study, an artificial RNA silencing inducible sequence (RSIS) was identified in rice (Oryza sativa). This sequence causes PTGS of 5′ or 3′ flanking-sequence-containing genes. Interestingly, two target genes can be simultaneously suppressed by linking a unique target sequence to either the 5′ or 3′ end of RSIS. Multiple gene suppression can be also achieved though a single transformation event by incorporating the multisite gateway system. Moreover, RSIS-mediated PTGS occurs in nuclei. Deep sequencing of small RNAs reveals that siRNAs are produced from RSIS-expressing cassettes and transitive siRNAs are produced from endogenous target genes. Furthermore, siRNAs are typically generated from untranscribed transgene terminator regions. The read-through transcripts from the RSIS-expression cassette were consistently observed, and most of these sequences were not polyadenylated. Collectively, this data indicates that RSIS inhibits proper transcription termination. The resulting transcripts are not polyadenylated. These transcripts containing RSIS become templates for double-stranded RNA synthesis in nuclei. This is followed by siRNA production and target degradation of target genes. PMID:22843666

  7. RNA silencing induced by an artificial sequence that prevents proper transcription termination in rice.

    PubMed

    Kawakatsu, Taiji; Wakasa, Yuhya; Yasuda, Hiroshi; Takaiwa, Fumio

    2012-10-01

    Posttranscriptional gene silencing (PTGS) is a sequence-specific mRNA degradation caused by small RNA, such as microRNA (miRNA) and small interfering RNA (siRNA). miRNAs are generated from MIRNA loci, whereas siRNAs originate from various sources of double-stranded RNA. In this study, an artificial RNA silencing inducible sequence (RSIS) was identified in rice (Oryza sativa). This sequence causes PTGS of 5' or 3' flanking-sequence-containing genes. Interestingly, two target genes can be simultaneously suppressed by linking a unique target sequence to either the 5' or 3' end of RSIS. Multiple gene suppression can be also achieved though a single transformation event by incorporating the multisite gateway system. Moreover, RSIS-mediated PTGS occurs in nuclei. Deep sequencing of small RNAs reveals that siRNAs are produced from RSIS-expressing cassettes and transitive siRNAs are produced from endogenous target genes. Furthermore, siRNAs are typically generated from untranscribed transgene terminator regions. The read-through transcripts from the RSIS-expression cassette were consistently observed, and most of these sequences were not polyadenylated. Collectively, this data indicates that RSIS inhibits proper transcription termination. The resulting transcripts are not polyadenylated. These transcripts containing RSIS become templates for double-stranded RNA synthesis in nuclei. This is followed by siRNA production and target degradation of target genes.

  8. Post-transcriptional regulation of meiotic genes by a nuclear RNA silencing complex

    PubMed Central

    Egan, Emily D.; Braun, Craig R.; Gygi, Steven P.; Moazed, Danesh

    2014-01-01

    RNA is a central component of gene-silencing pathways that regulate diverse cellular processes. In the fission yeast Schizosaccharomyces pombe, an RNA-based mechanism represses meiotic gene expression during vegetative growth. This pathway depends on the zinc finger protein Red1, which is required to degrade meiotic mRNAs as well as to target histone H3 lysine 9 (H3K9) methylation, a repressive chromatin mark, to a subset of meiotic genes. However, the mechanism of Red1 function is unknown. Here we use affinity purification and mass spectrometry to identify a Red1-containing nuclear RNA silencing (NURS) complex. In addition to Red1, this complex includes the Mtl1, Red5, Ars2, Rmn1, and Iss10 proteins and associates with several other complexes that are involved in either signaling or mediating RNA silencing. By analyzing the effects of gene knockouts and inducible knockdown alleles, we show that NURS subunits regulate RNA degradation and H3K9 methylation at meiotic genes. We also identify roles for individual NURS subunits in interactions with Mmi1, an RNA-binding protein that marks meiotic RNAs for destruction, and the nuclear exosome RNA degradation complex. Finally, we show that the levels of H3K9 methylation at meiotic genes are not sufficient to restrict RNA polymerase II access or repress gene expression during vegetative growth. Our results demonstrate that Red1 partners with other proteins to silence meiotic gene expression at the post-transcriptional level. Conservation of a NURS-like complex in human cells suggests that this pathway plays an ancient and fundamental role in RNA silencing. PMID:24713849

  9. A novel form of transcriptional silencing by Sum1-1 requires Hst1 and the origin recognition complex.

    PubMed

    Sutton, A; Heller, R C; Landry, J; Choy, J S; Sirko, A; Sternglanz, R

    2001-05-01

    In the yeast Saccharomyces cerevisiae, a and alpha mating-type information is stored in transcriptionally silenced cassettes called HML and HMR. Silencing of these loci, maintained by the formation of a specialized type of heterochromatin, requires trans-acting proteins and cis-acting elements. Proteins required for silencing include the Sir2 NAD(+)-dependent deacetylase, Sir3, and Sir4. Factors that bind to the cis elements at HMR and HML and that are important for silencing include the origin recognition complex (ORC). Mutations of any of these Sir proteins or combinations of cis elements result in loss of silencing. SUM1-1 was previously identified as a dominant mutation that restores silencing to HMR in the absence of either the Sir proteins or some of the cis elements. We have investigated the novel mechanism whereby Sum1-1 causes Sir-independent silencing at HMR and present the following findings: Sum1-1 requires the Sir2 homolog, Hst1, for silencing and most probably requires the NAD(+)-dependent deacetylase activity of this protein. Sum1-1 interacts strongly with ORC, and this strong interaction is dependent on HMR DNA. Furthermore, ORC is required for Sum1-1-mediated silencing at HMR. These observations lead to a model for Sum1-1 silencing of HMR in which Sum1-1 is recruited to HMR by binding to ORC. Sum1-1, in turn, recruits Hst1. Hst1 then deacetylates histones or other chromatin-associated proteins to cause chromatin condensation and transcriptional silencing.

  10. Bottlenecks in Development of Retinal Therapeutic Post-Transcriptional Gene Silencing Agents

    PubMed Central

    Sullivan, Jack M.; Yau, Edwin H.; Taggart, R. Thomas; Butler, Mark C.; Kolniak, Tiffany A.

    2011-01-01

    Development of post-transcriptional gene silencing (PTGS) agents for therapeutic purposes is an immense challenge in modern biology. Established technologies used to knockdown a specific target RNA and its cognate protein: antisense, ribozyme, RNAi, all conditionally depend upon an initial, critical annealing event of the PTGS ligand to a target RNA. In this review we address the nature of the bottlenecks, emphasizing the biocomplexity of target RNA structure, that currently limit PTGS therapeutic development. We briefly review existing and emerging technologies designed to release these constraints to realize the potential of PTGS agents in gene based therapies. PMID:17976683

  11. MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells

    PubMed Central

    Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A.; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

    2015-01-01

    RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression. PMID

  12. Effect of orientation of transcription of a gene in an inverted transferred DNA repeat on transcriptional gene silencing in rice transgenics-a case study.

    PubMed

    Ramkumar, Thakku R; Parameswari, Chidambaram; Sugapriya, Thennavan; Veluthambi, Karuppannan

    2015-01-01

    We studied transgene silencing in two transgenic rice plants, OSM25 and COT-OSM4, which harboured two different types of right border (RB)-centered inverted transferred DNA (T-DNA) repeats (IRs). The T-DNA in OSM25 has three genes gus, OSM and hph, all under the transcriptional control of the Cauliflower mosaic virus 35S promoter (P35S). The gus gene, which is proximal to the RB, is in a convergent orientation of transcription in the IR. OSM25 displayed silencing of all three transgenes. Nuclear run-on transcription analysis revealed that silencing of gus, OSM and hph genes in OSM25 operates at the transcriptional level. P35S showed hypermethylation in OSM25 plants. COT-OSM4 has P35S-driven gus and hph genes in its T-DNA. The hph gene, which is proximal to the RB, is in a divergent orientation of transcription in the IR. Unlike in OSM25, the transgenes in COT-OSM4 showed no silencing. These findings show that convergent orientation of transcription of a gene at the origin of an IR is important for transgene silencing.

  13. DNA Elements Reducing Transcriptional Gene Silencing Revealed by a Novel Screening Strategy

    PubMed Central

    Ueno, Keiichiro; Ohashi, Yuko; Mitsuhara, Ichiro

    2013-01-01

    Transcriptional gene silencing (TGS)–a phenomenon observed in endogenous genes/transgenes in eukaryotes–is a huge hindrance to transgenic technology and occurs mainly when the genes involved share sequence homology in their promoter regions. TGS depends on chromosomal position, suggesting the existence of genomic elements that suppress TGS. However, no systematic approach to identify such DNA elements has yet been reported. Here, we developed a successful novel screening strategy to identify such elements (anti-silencing regions–ASRs), based on their ability to protect a flanked transgene from TGS. A silenced transgenic tobacco plant in which a subsequently introduced transgene undergoes obligatory promoter-homology dependent TGS in trans allowed the ability of DNA elements to prevent TGS to be used as the screening criterion. We also identified ASRs in a genomic library from a different plant species (Lotus japonicus: a perennial legume); the ASRs include portions of Ty1/copia retrotransposon-like and pararetrovirus-like sequences; the retrotransposon-like sequences also showed interspecies anti-TGS activity in a TGS-induction system in Arabidopsis. Anti-TGS elements could provide effective tools to reduce TGS and ensure proper regulation of transgene expression. Furthermore, the screening strategy described here will also facilitate the efficient identification of new classes of anti-TGS elements. PMID:23382937

  14. Depletion of MOM1 in non-dividing cells of Arabidopsis plants releases transcriptional gene silencing.

    PubMed

    Tariq, Muhammad; Habu, Yoshiki; Paszkowski, Jerzy

    2002-10-01

    Mitotic and meiotic inheritance of epigenetic information is coupled to the reproduction of chromatin conformation and DNA methylation patterns. This implies that the S phase of the cell cycle provides a window of opportunity for changes in epigenetic determination. Recent studies, however, have suggested that chromatin structure is also rather dynamic in quiescent cells of multicellular eukaryotes and that silent heterochromatic regions can become accessible to transcription. Such epigenetic flexibility in differentiated tissues could be of physiological importance. The mechanisms and molecular components involved are of great interest but as yet unknown. We examined MOM1 (Morpheus' Molecule 1), a regulator of transcriptional gene silencing (TGS) that acts independently of DNA methylation, for its role in the maintenance of TGS in non-dividing, differentiated cells. The results provide evidence that TGS maintenance mediated by MOM1 is a dynamic process that can be modified in non-dividing cells of mature plant organs by depletion of MOM1.

  15. Identification of Multiple Proteins Coupling Transcriptional Gene Silencing to Genome Stability in Arabidopsis thaliana.

    PubMed

    Hale, Christopher J; Potok, Magdalena E; Lopez, Jennifer; Do, Truman; Liu, Ao; Gallego-Bartolome, Javier; Michaels, Scott D; Jacobsen, Steven E

    2016-06-01

    Eukaryotic genomes are regulated by epigenetic marks that act to modulate transcriptional control as well as to regulate DNA replication and repair. In Arabidopsis thaliana, mutation of the ATXR5 and ATXR6 histone methyltransferases causes reduction in histone H3 lysine 27 monomethylation, transcriptional upregulation of transposons, and a genome instability defect in which there is an accumulation of excess DNA corresponding to pericentromeric heterochromatin. We designed a forward genetic screen to identify suppressors of the atxr5/6 phenotype that uncovered loss-of-function mutations in two components of the TREX-2 complex (AtTHP1, AtSAC3B), a SUMO-interacting E3 ubiquitin ligase (AtSTUbL2) and a methyl-binding domain protein (AtMBD9). Additionally, using a reverse genetic approach, we show that a mutation in a plant homolog of the tumor suppressor gene BRCA1 enhances the atxr5/6 phenotype. Through characterization of these mutations, our results suggest models for the production atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell, and suggest that the atxr5 atxr6 transcriptional defects may be the cause of the genome instability defects in the mutants. These findings highlight the critical intersection of transcriptional silencing and DNA replication in the maintenance of genome stability of heterochromatin.

  16. Alleviating GAA Repeat Induced Transcriptional Silencing of the Friedreich's Ataxia Gene During Somatic Cell Reprogramming.

    PubMed

    Polak, Urszula; Li, Yanjie; Butler, Jill Sergesketter; Napierala, Marek

    2016-12-01

    Friedreich's ataxia (FRDA) is the most common autosomal recessive ataxia. This severe neurodegenerative disease is caused by an expansion of guanine-adenine-adenine (GAA) repeats located in the first intron of the frataxin (FXN) gene, which represses its transcription. Although transcriptional silencing is associated with heterochromatin-like changes in the vicinity of the expanded GAAs, the exact mechanism and pathways involved in transcriptional inhibition are largely unknown. As major remodeling of the epigenome is associated with somatic cell reprogramming, modulating chromatin modification pathways during the cellular transition from a somatic to a pluripotent state is likely to generate permanent changes to the epigenetic landscape. We hypothesize that the epigenetic modifications in the vicinity of the GAA repeats can be reversed by pharmacological modulation during somatic cell reprogramming. We reprogrammed FRDA fibroblasts into induced pluripotent stem cells (iPSCs) in the presence of various small molecules that target DNA methylation and histone acetylation and methylation. Treatment of FRDA iPSCs with two compounds, sodium butyrate (NaB) and Parnate, led to an increase in FXN expression and correction of repressive marks at the FXN locus, which persisted for several passages. However, prolonged culture of the epigenetically modified FRDA iPSCs led to progressive expansions of the GAA repeats and a corresponding decrease in FXN expression. Furthermore, we uncovered that differentiation of these iPSCs into neurons also results in resilencing of the FXN gene. Taken together, these results demonstrate that transcriptional repression caused by long GAA repeat tracts can be partially or transiently reversed by altering particular epigenetic modifications, thus revealing possibilities for detailed analyses of silencing mechanism and development of new therapeutic approaches for FRDA.

  17. Transcriptional Silencing of Moloney Murine Leukemia Virus in Human Embryonic Carcinoma Cells.

    PubMed

    Wang, Gary Z; Goff, Stephen P

    2017-01-01

    Embryonic carcinoma (EC) cells are malignant counterparts of embryonic stem (ES) cells and serve as useful models for investigating cellular differentiation and human embryogenesis. Though the susceptibility of murine EC cells to retroviral infection has been extensively analyzed, few studies of retrovirus infection of human EC cells have been performed. We tested the susceptibility of human EC cells to transduction by retroviral vectors derived from three different retroviral genera. We show that human EC cells efficiently express reporter genes delivered by vectors based on human immunodeficiency virus type 1 (HIV-1) and Mason-Pfizer monkey virus (M-PMV) but not Moloney murine leukemia virus (MLV). In human EC cells, MLV integration occurs normally, but no viral gene expression is observed. The block to MLV expression of MLV genomes is relieved upon cellular differentiation. The lack of gene expression is correlated with transcriptional silencing of the MLV promoter through the deposition of repressive histone marks as well as DNA methylation. Moreover, depletion of SETDB1, a histone methyltransferase, resulted in a loss of transcriptional silencing and upregulation of MLV gene expression. Finally, we provide evidence showing that the lack of MLV gene expression may be attributed in part to the lack of MLV enhancer function in human EC cells.

  18. A novel type of silencing factor, Clr2, is necessary for transcriptional silencing at various chromosomal locations in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Bjerling, Pernilla; Ekwall, Karl; Egel, Richard; Thon, Geneviève

    2004-01-01

    The mating-type region of the fission yeast Schizosaccharomyces pombe comprises three loci: mat1, mat2-P and mat3-M. mat1 is expressed and determines the mating type of the cell. mat2-P and mat3-M are two storage cassettes located in a 17 kb heterochromatic region with features identical to those of mammalian heterochromatin. Mutations in the swi6+, clr1+, clr2+, clr3+, clr4+ and clr6+ genes were obtained in screens for factors necessary for silencing the mat2-P-mat3-M region. swi6+ encodes a chromodomain protein, clr3+ and clr6+ histone deacetylases, and clr4+ a histone methyltransferase. Here, we describe the cloning and characterization of clr2+. The clr2+ gene encodes a 62 kDa protein with no obvious sequence homologs. Deletion of clr2+ not only affects transcriptional repression in the mating-type region, but also centromeric silencing and silencing of a PolII-transcribed gene inserted in the rDNA repeats. Using chromatin immunoprecipitation, we show that Clr2 is necessary for histone hypoacetylation in the mating-type region, suggesting that Clr2 acts upstream of histone deacetylases to promote transcriptional silencing.

  19. The human involucrin gene is transcriptionally repressed through a tissue-specific silencer element recognized by Oct-2.

    PubMed

    Azuara-Liceaga, Elisa; Sandoval, Marisol; Corona, Matilde; Gariglio, Patricio; López-Bayghen, Esther

    2004-05-28

    Involucrin is an important marker of epithelial differentiation which expression is upregulated just after basal cells are pushed into the suprabasal layer in stratified epithelia. Several transcription factors and regulatory elements had been described as responsible for turning on the gene. However, it is evident that in basal cell layer, additional mechanisms are involved in keeping the gene silent before the differentiation process starts. In this work, we located a potential transcriptional silencer in a 52bp sequence whose integrity is necessary for silencing the proximal enhancer promoter element (PEP) in multiplying keratinocytes. Octamer-binding sites were noticed in this fragment and the specific binding of Oct-2 transcription factor was detected. Oct-2 appears to be implicated in an epithelial-specific repression activity recorded only in keratinocytes and C33-A cell line. Overexpression of Oct-2 repressed the involucrin promoter activity in epithelial cells and in the presence of the silencer element.

  20. Histone modifications silence the GATA transcription factor genes in ovarian cancer.

    PubMed

    Caslini, C; Capo-chichi, C D; Roland, I H; Nicolas, E; Yeung, A T; Xu, X-X

    2006-08-31

    Altered expression of GATA factors was found and proposed as the underlying mechanism for dedifferentiation in ovarian carcinogenesis. In particular, GATA6 is lost or excluded from the nucleus in 85% of ovarian tumors and GATA4 expression is absent in majority of ovarian cancer cell lines. Here, we evaluated their DNA and histone epigenetic modifications in five ovarian epithelial and carcinoma cell lines (human 'immortalized' ovarian surface epithelium (HIO)-117, HIO-114, A2780, SKOV3 and ES2). GATA4 and GATA6 gene silencing was found to correlate with hypoacetylation of histones H3 and H4 and loss of histone H3/lysine K4 tri-methylation at their promoters in all lines. Conversely, histone H3/lysine K9 di-methylation and HP1gamma association were not observed, excluding reorganization of GATA genes into heterochromatic structures. The histone deacetylase inhibitor trichostatin A, but not the DNA methylation inhibitor 5'-aza-2'-deoxycytidine, re-established the expression of GATA4 and/or GATA6 in A2780 and HIO-114 cells, correlating with increased histone H3 and H4 acetylation, histone H3 lysine K4 methylation and DNase I sensitivity at the promoters. Therefore, altered histone modification of the promoter loci is one mechanism responsible for the silencing of GATA transcription factors and the subsequent loss of a target gene, the tumor suppressor Disabled-2, in ovarian carcinogenesis.

  1. Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment.

    PubMed

    Le, Huy Quang; Ghatak, Sushmita; Yeung, Ching-Yan Chloé; Tellkamp, Frederik; Günschmann, Christian; Dieterich, Christoph; Yeroslaviz, Assa; Habermann, Bianca; Pombo, Ana; Niessen, Carien M; Wickström, Sara A

    2016-08-01

    Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. We show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. Our results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis.

  2. [The analysis of rbcS gene function by post-transcription gene silencing in Nicotiana benthamiana].

    PubMed

    Zhou, Xiao-Fu; Ma, Peng-Da; Wang, Ren-Hou; Zhu, Xiao-Juan; Liu, Bao; Wang, Xing-Zhi

    2005-06-01

    A system of virus-induced post-transcriptional gene silencing for studying rbcS gene function was established and optimized using tobacco rattle virus vector and Nicotiana benthamiana as experimental materiaes. The following analyses were conducted: phenotypic characterization of rbcS gene silenced plants, transcription levels of rbcS gene by RT-PCR; protein levels of rbcS by the antibodies of rbcS and rbcL and photosynthetic pigments wntents in rbcS silenced plants by HPLC method. The results showed that the seedlings at 21-24-day-old and Agrobacterium concentration at OD600 = 1-1.5 gave the best results for gene silencing. The expression level of rbcL was very likely regulated by rbcS, and rbcS gene did not relate to the collection of photosynthetic energy. Probability analysis showed that the tobacco rattle virus vector system is a useful and effective technique to study rbcS gene function via post-transcriptional gene silencing.

  3. CENP-B Cooperates with Set1 in Bidirectional Transcriptional Silencing and Genome Organization of Retrotransposons

    PubMed Central

    Lorenz, David R.; Mikheyeva, Irina V.; Johansen, Peter; Meyer, Lauren; Berg, Anastasia; Grewal, Shiv I. S.

    2012-01-01

    Regulation of transposable elements (TEs) is critical to the integrity of the host genome. The fission yeast Schizosaccharomyces pombe homologs of mammalian CENP-B perform a host genome surveillance role by controlling Tf2 long terminal repeat (LTR) retrotransposons. However, the mechanisms by which CENP-Bs effect their functions are ill defined. Here, we show that the multifaceted roles of Abp1, the prominent member of fission yeast CENP-Bs, are mediated in part via recognition of a 10-bp AT-rich motif present in most LTRs and require the DNA-binding, transposase, and dimerization domains of Abp1 to maintain transcriptional repression and genome organization. Expression profiling analyses indicated that Abp1 recruits class I/II histone deacetylases (HDACs) to repress Tf2 retrotransposons and genes activated in response to stresses. We demonstrate that class I/II HDACs and sirtuins mediate the clustering of dispersed Tf2 retrotransposons into Tf bodies. Intriguingly, we uncovered an unexpected cooperation between Abp1 and the histone H3K4 methyltransferase Set1 in regulating sense and antisense transcriptional silencing of Tf2 retrotransposons and Tf body integrity. Moreover, Set1-mediated regulation of Tf2 expression and nuclear organization appears to be largely independent of H3K4 methylation. Our study illuminates a molecular pathway involving a transposase-containing transcription factor that cooperates with chromatin modifiers to regulate TE activities. PMID:22907751

  4. Transcriptional silencing of heterologous anther promoters in maize: a genetic method to replace detasseling for seed production.

    PubMed

    Cigan, A Mark; Haug-Collet, Kristin; Clapp, Joshua

    2014-09-01

    The promoter of the maize male fertility gene ZmMs45, and other anther-specific maize promoters, was previously shown to be transcriptionally silenced by constitutively expressed promoter-inverted repeat RNAs (pIRs). In addition, ZmMS45pIR-mediated male sterility was reversed by co-expression of Ms45 transcribed by promoters not targeted by pIR RNA silencing. In this report, male fertility was restored to ms45 maize by fusing non-maize inflorescence promoters to the ZmMS45 coding region. This complementation assay also established that these rice or Arabidopsis promoters, when expressed as pIRs, functioned to silence sequence identical promoters. These observations were exploited to develop a genetic method to replace maize detasseling during hybrid seed production. In this system, the ZmMS45 coding region was fused to one of two dissimilar non-maize promoters to generate paired sets of ms45 recessive inbred parents which could be self-pollinated and maintained independently. Linked to each unique Ms45 gene was a non-maize pIR which targeted the promoter transcribing the Ms45 copy contained in the paired inbred parent plant. A cross of these pairs brings the dissimilar pIR cassettes together and resulted in silencing both transformed copies of Ms45. The net result uncovers the ms45 allele carried by the inbreds yielding male sterile progeny. The application of heterologous promoters and transcriptional silencing in plants provides an alternative to post-transcriptional gene silencing as a means to restore and silence gene function in plants.

  5. Post-Transcriptional Silencing of Flavonol Synthase mRNA in Tobacco Leads to Fruits with Arrested Seed Set

    PubMed Central

    Mahajan, Monika; Ahuja, Paramvir Singh; Yadav, Sudesh Kumar

    2011-01-01

    Flavonoids are synthesized by phenylpropanoid pathway. They are known to participate in large number of physiological and biochemical processes in plants. Parthenocarpy and male sterility has earlier been reported by silencing chalcone synthase (CHS) encoding gene. Silencing of CHS has blocked the synthesis of most of useful flavonoids including flavan-3-ols and flavonols. Also, these studies could not identify whether parthenocarpy/male sterility were due to lack of flavan-3-ols or flavonols or both. Flavonol synthase (FLS) is an important enzyme of flavonoid pathway that catalyzes the formation of flavonols. In this article, we propose a novel strategy towards the generation of seedless or less-seeded fruits by downregulation of flavonol biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) through post-transcriptional gene silencing (PTGS) of FLS encoding mRNA. The FLS silenced lines were observed for 20-80% reduction in FLS encoding gene expression and 25–93% reduction in flavonol (quercetin) content. Interestingly, these FLS silenced tobacco lines also showed reduction in their anthocyanidins content. While the content of flavan-3-ols (catechin, epi-catechin and epi-gallocatechin) was found to be increased in FLS silenced lines. The delayed flowering in FLS silenced lines could be due to decrease in level of indole acetic acid (IAA) at apical region of their shoots. Furthermore, the pollen germination was hampered and pollens were unable to produce functional pollen tube in FLS silenced tobacco lines. Pods of FLS silenced lines contained significantly less number of seeds. The in vitro and in vivo studies where 1 µM quercetin was supplied to germination media, documented the restoration of normal pollen germination and pollen tube growth. This finding identified the role of flavonols particularly quercetin in pollen germination as well as in the regulation of plant fertility. Results also suggest a novel approach towards generation of seedless

  6. A role for DNA polymerase α in epigenetic control of transcriptional silencing in fission yeast

    PubMed Central

    Nakayama, Jun-ichi; Allshire, Robin C.; Klar, Amar J.S.; Grewal, Shiv I.S.

    2001-01-01

    In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase α (polα) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that polα mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polα and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Polα in vitro. Moreover, silencing-defective mutant Polα displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Polα, in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  7. Human immunodeficiency virus type 1 negative factor is a transcriptional silencer.

    PubMed

    Niederman, T M; Thielan, B J; Ratner, L

    1989-02-01

    The negative factor (nef) of human immunodeficiency virus (HIV) type 1 acts to down-regulate virus replication. To decipher the step in the virus life cycle affected by nef, functional proviral clones with (pHIV F-) or without (pHIV F+) a deletion mutation in the nef gene were constructed. In CD4+ cells, 30- to 50-fold more virus was produced over the course of 18-20 days with cultures infected with F- compared to F+ virus. In CD4- cell lines, 2- to 10-fold greater virus production was found from cultures transfected with pHIV F- than those transfected with pHIV F+. The negative regulatory effects of nef on pHIV F- could be supplied in trans with a plasmid expressing only the nef gene product. Virus produced by COS-1 cells transfected with pHIV F- or pHIV F+ showed similar binding, uptake, uncoating, and reverse transcription. Analysis of HIV-1 RNA and structural protein levels and rates of viral RNA synthesis in CD4- cells also showed 2- to 10-fold higher levels in cells transfected with pHIV F- compared to pHIV F+. The activity of a HIV-1-chloramphenicol acetyltransferase (CAT) plasmid was also suppressed by nef, whereas other CAT plasmids were unaffected. These findings demonstrate that nef acts as a specific silencer of HIV-1 transcription. This activity may be critical for maintenance of HIV-1 latency in vivo.

  8. Transcription factor Sp3 is silenced through SUMO modification by PIAS1

    PubMed Central

    Sapetschnig, Alexandra; Rischitor, Grigore; Braun, Harald; Doll, Andreas; Schergaut, Marion; Melchior, Frauke; Suske, Guntram

    2002-01-01

    Sp3 is a ubiquitous transcription factor closely related to Sp1. Here we show that Sp3 is a target for SUMO modification in vivo and in vitro. SUMO modification of Sp3 occurs at a single lysine located between the second glutamine-rich activation domain and the DNA-binding domain. Mutational analyses identified the sequence IKXE as essential for SUMO conjugation to Sp3. We identified the protein inhibitor of activated STAT1 (PIAS1) as an interaction partner of Sp3 and Ubc9. Moreover, PIAS1 strongly stimulated SUMO conjugation to Sp3, thus acting as an E3 ligase for SUMO conjugation to Sp3. All mutations that prevented SUMO modification in vitro strongly enhanced the transcriptional activity of Sp3, showing that SUMO modification silences Sp3 activity. SUMO-modified Sp3 bound to DNA with similar specificity and affinity as unmodified Sp3. However, DNA-bound Sp3 did not act as a substrate for SUMO modification. PMID:12356736

  9. Evidence That the Transcriptional Regulators Sin3 and Rpd3, and a Novel Gene (Sds3) with Similar Functions, Are Involved in Transcriptional Silencing in S. Cerevisiae

    PubMed Central

    Vannier, D.; Balderes, D.; Shore, D.

    1996-01-01

    In a screen for extragenic suppressors of a silencing defective rap1(s) hmrΔA strain, recessive mutations in 21 different genes were found that restored repression to HMR. We describe the characterization of three of these SDS (suppressors of defective silencing) genes. SDS16 and SDS6 are known transcriptional modifiers, SIN3(RPD1/UME4/SDI1/GAM2) and RPD3(SDI2), respectively, while the third is a novel gene, SDS3. SDS3 shares the meiotic functions of SIN3 and RPD3 in that it represses IME2 in haploid cells and is necessary for sporulation in diploid cells. However, sds3 mutations differ from sin3 and rpd3 mutations in that they do not derepress TRK2. These sds mutations suppress a variety of cis- and trans-defects, which impair the establishment of silencing at HMR. Any one of the sds mutations slightly increases telomere position effect while a striking synergistic increase in repression is observed in a rap1(s) background. Epistasis studies suggest that SDS3 works in a different pathway from RPD3 and SIN3 to affect silencing at HMR. Together these results show that defects in certain general transcriptional modifiers can have a pronounced influence on position-effect gene silencing in yeast. Mechanisms for this increase in postion effect are discussed. PMID:8978024

  10. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots

    PubMed Central

    Rizvi, Noreen F.; Weaver, Jessica D.; Cram, Erin J.; Lee-Parsons, Carolyn W. T.

    2016-01-01

    The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis. PMID:27467510

  11. Post-transcriptional gene silencing of the p23 silencing suppressor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime.

    PubMed

    Fagoaga, Carmen; López, Carmelo; de Mendoza, Alfonso Hermoso; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Peña, Leandro

    2006-01-01

    Previously, we have shown that most Mexican limes (Citrus aurantifolia (Christ.) Swing.) expressing the p23 gene of Citrus tristeza virus (CTV) exhibit aberrations resembling viral leaf symptoms. Here we report that five independent transgenic lines having normal phenotype displayed characteristics typical of post-transcriptional gene silencing (PTGS): multiple copies of the transgene, low levels of the corresponding mRNA, methylation of the silenced transgene, and accumulation of p23-specific small interfering RNAs (siRNAs). When graft- or aphid-inoculated with CTV, some propagations of these silenced lines were immune: they neither expressed symptoms nor accumulated virions and viral RNA as estimated by DAS-ELISA and Northern blot hybridization, respectively. Other propagations were moderately resistant because they became infected later and showed attenuated symptoms compared to controls. The susceptible propagations, in addition to symptom expression and elevated virus titer, accumulated p23-specific siRNAs at levels significantly higher than immune or non-inoculated propagations, and showed transgene demethylation. This variable response among clonal transformants indicates that factors other than the genetic background of the transgenic plants play a key role in PTGS-mediated resistance.

  12. Distinctive profiles of small RNA couple inverted repeat-induced post-transcriptional gene silencing with endogenous RNA silencing pathways in Arabidopsis

    PubMed Central

    Matvienko, Marta; Piskurewicz, Urszula; Xu, Huaqin; Martineau, Belinda; Wong, Joan; Govindarajulu, Manjula; Kozik, Alexander; Michelmore, Richard W.

    2014-01-01

    The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of post-transcriptional gene silencing (PTGS) and determine its specificity. Despite its application in agriculture and broad utility in plant research, the mechanism of IR-PTGS is incompletely understood. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE SYNTHASE (At-CHS) gene fragments. Levels of PTGS were found to depend on the orientation and position of the fragment in the IR construct. Deep sequencing and mapping of sRNAs to corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation that revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. Detailed analyses of poly-A cleavage products from At-CHS mRNA confirmed this hypothesis. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes. PMID:25344399

  13. Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer

    PubMed Central

    Jägle, Sabine; Rönsch, Kerstin; Timme, Sylvia; Andrlová, Hana; Bertrand, Miriam; Jäger, Marcel; Proske, Amelie; Schrempp, Monika; Yousaf, Afsheen; Michoel, Tom; Zeiser, Robert; Werner, Martin; Lassmann, Silke; Hecht, Andreas

    2014-01-01

    The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma–carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/β-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer. PMID:24707046

  14. Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer.

    PubMed

    Jägle, Sabine; Rönsch, Kerstin; Timme, Sylvia; Andrlová, Hana; Bertrand, Miriam; Jäger, Marcel; Proske, Amelie; Schrempp, Monika; Yousaf, Afsheen; Michoel, Tom; Zeiser, Robert; Werner, Martin; Lassmann, Silke; Hecht, Andreas

    2014-04-01

    The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma-carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/β-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer.

  15. The gene silencing transcription factor REST represses miR-132 expression in hippocampal neurons destined to die

    PubMed Central

    Hwang, Jee-Yeon; Kaneko, Naoki; Noh, Kyung-Min; Pontarelli, Fabrizio; Zukin, R. Suzanne

    2014-01-01

    The gene silencing transcription factor REST/NRSF (Repressor Element-1 (RE1) Silencing Transcription Factor/Neuron-Restrictive Silencer Factor) actively represses a large array of coding and noncoding neuron-specific genes important to synaptic plasticity including miR-132. miR-132 is a neuron-specific microRNA and plays a pivotal role in synaptogenesis, synaptic plasticity and structural remodeling. However, a role for miR-132 in neuronal death is not, as yet, well-delineated. Here we show that ischemic insults promote REST binding and epigenetic remodeling at the miR-132 promoter and silencing of miR-132 expression in selectively-vulnerable hippocampal CA1 neurons. REST occupancy was not altered at the miR-9 or miR-124a promoters despite the presence of RE1 sites, indicating REST target specificity. Ischemia induced a substantial decrease in two marks of active gene transcription, dimethylation of lysine 4 on core histone 3 (H3K4me2) and acetylation of lysine 9 on H3 (H3K9ac) at the miR-132 promoter. RNAi-mediated depletion of REST in vivo blocked ischemia-induced loss of miR-132 in insulted hippocampal neurons, consistent with a causal relation between activation of REST and silencing of miR-132. Overexpression of miR-132 in primary cultures of hippocampal neurons or delivered directly into the CA1 of living rats by means of the lentiviral expression system prior to induction of ischemia afforded robust protection against ischemia-induced neuronal death. These findings document a previously unappreciated role for REST-dependent repression of miR-132 in the neuronal death associated with global ischemia and identify a novel therapeutic target for amelioration of the neurodegeneration and cognitive deficits associated with ischemic stroke. PMID:25108103

  16. Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors.

    PubMed

    Dulmovits, Brian M; Appiah-Kubi, Abena O; Papoin, Julien; Hale, John; He, Mingzhu; Al-Abed, Yousef; Didier, Sebastien; Gould, Michael; Husain-Krautter, Sehba; Singh, Sharon A; Chan, Kyle W H; Vlachos, Adrianna; Allen, Steven L; Taylor, Naomi; Marambaud, Philippe; An, Xiuli; Gallagher, Patrick G; Mohandas, Narla; Lipton, Jeffrey M; Liu, Johnson M; Blanc, Lionel

    2016-03-17

    Current therapeutic strategies for sickle cell anemia are aimed at reactivating fetal hemoglobin. Pomalidomide, a third-generation immunomodulatory drug, was proposed to induce fetal hemoglobin production by an unknown mechanism. Here, we report that pomalidomide induced a fetal-like erythroid differentiation program, leading to a reversion of γ-globin silencing in adult human erythroblasts. Pomalidomide acted early by transiently delaying erythropoiesis at the burst-forming unit-erythroid/colony-forming unit-erythroid transition, but without affecting terminal differentiation. Further, the transcription networks involved in γ-globin repression were selectively and differentially affected by pomalidomide including BCL11A, SOX6, IKZF1, KLF1, and LSD1. IKAROS (IKZF1), a known target of pomalidomide, was degraded by the proteasome, but was not the key effector of this program, because genetic ablation of IKZF1 did not phenocopy pomalidomide treatment. Notably, the pomalidomide-induced reprogramming was conserved in hematopoietic progenitors from individuals with sickle cell anemia. Moreover, multiple myeloma patients treated with pomalidomide demonstrated increased in vivo γ-globin levels in their erythrocytes. Together, these data reveal the molecular mechanisms by which pomalidomide reactivates fetal hemoglobin, reinforcing its potential as a treatment for patients with β-hemoglobinopathies.

  17. Variables and Strategies in Development of Therapeutic Post-Transcriptional Gene Silencing Agents

    PubMed Central

    Sullivan, Jack M.; Yau, Edwin H.; Kolniak, Tiffany A.; Sheflin, Lowell G.; Taggart, R. Thomas; Abdelmaksoud, Heba E.

    2011-01-01

    Post-transcriptional gene silencing (PTGS) agents such as ribozymes, RNAi and antisense have substantial potential for gene therapy of human retinal degenerations. These technologies are used to knockdown a specific target RNA and its cognate protein. The disease target mRNA may be a mutant mRNA causing an autosomal dominant retinal degeneration or a normal mRNA that is overexpressed in certain diseases. All PTGS technologies depend upon the initial critical annealing event of the PTGS ligand to the target RNA. This event requires that the PTGS agent is in a conformational state able to support hybridization and that the target have a large and accessible single-stranded platform to allow rapid annealing, although such platforms are rare. We address the biocomplexity that currently limits PTGS therapeutic development with particular emphasis on biophysical variables that influence cellular performance. We address the different strategies that can be used for development of PTGS agents intended for therapeutic translation. These issues apply generally to the development of PTGS agents for retinal, ocular, or systemic diseases. This review should assist the interested reader to rapidly appreciate critical variables in PTGS development and facilitate initial design and testing of such agents against new targets of clinical interest. PMID:21785698

  18. Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids.

    PubMed

    Hsieh, Ming-Hsien; Pan, Zhao-Jun; Lai, Pei-Han; Lu, Hsiang-Chia; Yeh, Hsin-Hung; Hsu, Chia-Chi; Wu, Wan-Lin; Chung, Mei-Chu; Wang, Shyh-Shyan; Chen, Wen-Huei; Chen, Hong-Hwa

    2013-09-01

    Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis.

  19. Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids

    PubMed Central

    Hsieh, Ming-Hsien; Pan, Zhao-Jun; Lai, Pei-Han; Lu, Hsiang-Chia; Yeh, Hsin-Hung; Hsu, Chia-Chi; Wu, Wan-Lin; Chung, Mei-Chu; Wang, Shyh-Shyan; Chen, Wen-Huei; Chen, Hong-Hwa

    2013-01-01

    Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis. PMID:23956416

  20. Identification and gene-silencing of a putative odorant receptor transcription factor in Varroa destructor: possible role in olfaction.

    PubMed

    Singh, N K; Eliash, N; Stein, I; Kamer, Y; Ilia, Z; Rafaeli, A; Soroker, V

    2016-04-01

    The ectoparasitic mite Varroa destructor is one of the major threats to apiculture. Using a behavioural choice bioassay, we determined that phoretic mites were more successful in reaching a bee than reproductive mites, suggesting an energy trade-off between reproduction and host selection. We used both chemo-ecological and molecular strategies to identify the regulation of the olfactory machinery of Varroa and its association with reproduction. We focused on transcription regulation. Using primers designed to the conserved DNA binding region of transcription factors, we identified a gene transcript in V. destructor homologous to the pheromone receptor transcription factor (PRTF) gene of Pediculus humanus corporis. Quantitative PCR (qPCR) revealed that this PRTF-like gene transcript is expressed in the forelegs at higher levels than in the body devoid of forelegs. Subsequent comparative qPCR analysis showed that transcript expression was significantly higher in the phoretic as compared to the reproductive stage. Electrophysiological and behavioural studies revealed a reduction in the sensitivity of PRTF RNA interference-silenced mites to bee headspace, consistent with a reduction in the mites' ability to reach a host. In addition, vitellogenin expression was stimulated in PRTF-silenced mites to similar levels as found in reproductive mites. These data shed light upon the regulatory mechanism of host chemosensing in V. destructor. © 2016 The Royal Entomological Society.

  1. DmGTSF1 is necessary for Piwi–piRISC-mediated transcriptional transposon silencing in the Drosophila ovary

    PubMed Central

    Ohtani, Hitoshi; Iwasaki, Yuka W.; Shibuya, Aoi; Siomi, Haruhiko; Siomi, Mikiko C.; Saito, Kuniaki

    2013-01-01

    The Piwi–piRNA (PIWI-interacting RNA) complex (Piwi–piRISC) in Drosophila ovarian somatic cells represses transposons transcriptionally to maintain genome integrity; however, the underlying mechanisms remain obscure. Here, we reveal that DmGTSF1, a Drosophila homolog of gametocyte-specific factor 1 (GTSF1) (which is required for transposon silencing in mouse testes), is necessary for Piwi–piRISC to repress target transposons and neighboring genes. DmGTSF1 depletion affected neither piRNA biogenesis nor nuclear import of Piwi–piRISC. DmGTSF1 mutations caused derepression of transposons and loss of ovary follicle layers, resulting in female infertility. We suggest that DmGTSF1, a nuclear Piwi interactor, is an integral factor in Piwi–piRISC-mediated transcriptional silencing. PMID:23913921

  2. Sense transgene-induced post-transcriptional gene silencing in tobacco compromises the splicing of endogenous counterpart genes.

    PubMed

    Shin, Mi-Rae; Natsuume, Masaya; Matsumoto, Takashi; Hanaoka, Mitsumasa; Imai, Misaki; Iijima, Ken; Oka, Shin-Ichiro; Adachi, Eri; Kodama, Hiroaki

    2014-01-01

    Sense transgene-induced post-transcriptional gene silencing (S-PTGS) is thought to be a type of RNA silencing in which ARGONAUTE1 directs the small interfering RNA (siRNA)-mediated cleavage of a target mRNA in the cytoplasm. Here, we report that the altered splicing of endogenous counterpart genes is a main cause for the reduction of their mature mRNA levels. After the S-PTGS of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) gene, 3'-truncated, polyadenylated endo-NtFAD3 transcripts and 5'-truncated, intron-containing endo-NtFAD3 transcripts were detected in the total RNA fraction. Although transcription proceeded until the last exon of the endogenous NtFAD3 gene, intron-containing NtFAD3 transcripts accumulated in the nucleus of the S-PTGS plants. Several intron-containing NtFAD3 transcripts harboring most of the exon sequences were generated when an endogenous silencing suppressor gene, rgs-CaM, was overexpressed in the S-PTGS plants. These intron-containing NtFAD3 splice variants were generated in the presence of NtFAD3 siRNAs that are homologous to the nucleotide sequences of these splice variants. The results of this study indicate that the inhibition of endo-NtFAD3 gene expression is primarily directed via the alteration of splicing and not by cytoplasmic slicer activity. Our results suggest that the transgene and intron-containing endogenous counterpart genes are differentially suppressed in S-PTGS plants.

  3. The fail-safe mechanism of post-transcriptional silencing of unspliced HAC1 mRNA

    PubMed Central

    Di Santo, Rachael; Aboulhouda, Soufiane; Weinberg, David E

    2016-01-01

    HAC1 encodes a transcription factor that is the central effector of the unfolded protein response (UPR) in budding yeast. When the UPR is inactive, HAC1 mRNA is stored as an unspliced isoform in the cytoplasm and no Hac1 protein is detectable. Intron removal is both necessary and sufficient to relieve the post-transcriptional silencing of HAC1 mRNA, yet the precise mechanism by which the intron prevents Hac1 protein accumulation has remained elusive. Here, we show that a combination of inhibited translation initiation and accelerated protein degradation—both dependent on the intron—prevents the accumulation of Hac1 protein when the UPR is inactive. Functionally, both components of this fail-safe silencing mechanism are required to prevent ectopic production of Hac1 protein and concomitant activation of the UPR. Our results provide a mechanistic understanding of HAC1 regulation and reveal a novel strategy for complete post-transcriptional silencing of a cytoplasmic mRNA. DOI: http://dx.doi.org/10.7554/eLife.20069.001 PMID:27692069

  4. MOM1 and Pol-IV/V interactions regulate the intensity and specificity of transcriptional gene silencing

    PubMed Central

    Yokthongwattana, Chotika; Bucher, Etienne; Čaikovski, Marian; Vaillant, Isabelle; Nicolet, Joël; Scheid, Ortrun Mittelsten; Paszkowski, Jerzy

    2010-01-01

    It is commonly observed that onset or release of transcriptional gene silencing (TGS) correlates with alteration of repressive epigenetic marks. The TGS regulator MOM1 in Arabidopsis is exceptional since it regulates transcription in intermediate heterochromatin with only minor changes in epigenetic marks. We have isolated an enhancer of the mom1 mutation that points towards regulatory interplay between MOM1 and RNA polymerase-V (Pol-V). Pol-V transcribes heterochromatic loci, which seems to be required for maintenance of their silencing; however, it is still not clear how Pol-V is targeted to heterochromatin. We now provide evidence that Pol-V is required for MOM1-mediated suppression of transcription at a subset of its chromosomal targets. Thus, Pol-V genetically interacts with MOM1 in the control of gene silencing. Interestingly, functional cooperation of MOM1 and Pol-V not only broadens the range of the controlled loci in comparison to each individual factor, but also determines the degree of TGS. PMID:19910926

  5. Sequences throughout the basic beta-1,3-glucanase mRNA coding region are targets for homology dependent post-transcriptional gene silencing.

    PubMed

    Jacobs; Sanders; Bots; Andriessen; Van Eldik GJ; Litière; Van Montagu M; Cornelissen

    1999-10-01

    In the transgenic tobacco line T17, plants homozygous for the gn1 transgene display developmentally regulated post-transcriptional silencing of basic beta-1,3-glucanase genes. Previously, it has been shown that silencing involves a markedly increased turnover of silencing-target glucanase mRNAs. Using a two-component viral reporter system facilitated a comparison, in a quantitat- ive manner, of the relative silencing efficiencies of various sequences derived from the gn1 transgene. The results show that target sites for the silencing mechanism are present throughout the coding region of the gn1 mRNA. Similar-sized coding region sequences along the entire gn1 mRNA display a similar susceptibility to the silencing mechanism. The susceptibility to silencing increases as the coding region elements increase in size. Relative to internal sequences, the 5' and 3' terminal regions of the gn1 mRNA are inefficient targets for the silencing machinery. Importantly, sequences of the gn1 transgene that are not part of the mature gn1 mRNA are not recognized by the silencing machinery when expressed in chimeric viral RNAs. These results show that the glucanase silencing mechanism in T17 plants is primarily directed against gn1 mRNA-internal sequences and that terminal sequences of the gn1 mRNA are relatively unaffected by the silencing mechanism.

  6. Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing

    PubMed Central

    Kon, Tatsuya; Yoshikawa, Nobuyuki

    2014-01-01

    Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification. PMID:25426109

  7. Silencing of β1 integrin regulates airway remodeling by regulating the transcription of SOCE‑associated genes in asthmatic mice.

    PubMed

    Qiu, Chen; Liu, Wenwen; Shi, Fei; Fen, Mengjie; Ren, Lili; Qi, Hui

    2017-09-01

    The incidence of asthma is increasing globally; however, current treatments are only able to cure a certain proportion of patients. There is an urgent need to develop novel therapies. β1 integrin serves a role in the pathophysiology of asthma through the development of airway remodeling. The aim of the present study was to investigate silencing of the β1 integrin gene in pre‑clinical models of allergic asthma. BALB/c mice were sensitized with ovalbumin through intraperitoneal injection and repeated aerosolized ovalbumin. A short hairpin RNA of the β1 integrin gene was designed and transfected into mouse models of asthma in vivo, in order to evaluate whether silencing of the β1 integrin gene affects airway smooth muscle cell proliferation and inflammation by regulating the mRNA expression of store‑operated Ca2+ entry (SOCE)‑associated genes. Silencing the β1 integrin gene may downregulate β1 integrin mRNA while not statistically decreasing α‑smooth muscle actin gene expression and airway smooth muscle thickness. β1 integrin silencing was able to downregulate the transcription of SOCE‑associated genes to normal levels, including calcium release‑activated calcium modulator 1 and short transient receptor potential channel member 1, but not stromal interaction molecule 1, in asthma. Silencing of the β1 integrin gene additionally maintained nuclear factor of activated T‑cells cytoplasmic 1 gene expression, and inflammatory cytokines interleukin‑4 and interferon‑γ at normal levels. The results of the present study provide evidence to suggest that silencing of the β1 integrin gene may be of therapeutic benefit for patients with asthma.

  8. Manipulation of DET1 expression in tomato results in photomorphogenic phenotypes caused by post-transcriptional gene silencing

    PubMed Central

    Davuluri, Ganga Rao; van Tuinen, Ageeth; Mustilli, Anna Chiara; Manfredonia, Alessandro; Newman, Robert; Burgess, Diane; Brummell, David A.; King, Stephen R.; Palys, Joe; Uhlig, John; Pennings, Henk M. J.; Bowler, Chris

    2013-01-01

    Summary The tomato HIGH PIGMENT-2 gene encodes an orthologue of the Arabidopsis nuclear protein DE-ETIOLATED 1 (DET1). From genetic analyses it has been proposed that DET1 is a negative regulator of light signal transduction, and recent results indicate that it may control light-regulated gene expression at the level of chromatin remodelling. To gain further understanding about the function of DET1 during plant development, we generated a range of overexpression constructs and introduced them into tomato. Unexpectedly, we only observed phenotypes characteristic of DET1 inactivation, i.e. hyper-responsiveness to light. Molecular analysis indicated in all cases that these phenotypes were a result of suppression of endogenous DET1 expression, due to post-transcriptional gene silencing. DET1 silencing was often lethal when it occurred at relatively early stages of plant development, whereas light hyper-responsive phenotypes were obtained when silencing occurred later on. The appearance of phenotypes correlated with the generation of siRNAs but not DNA hypermethylation, and was most efficient when using constructs with mutations in the DET1 coding sequence or with constructs containing only the 3′-terminal portion of the gene. These results indicate an important function for DET1 throughout plant development and demonstrate that silencing of DET1 in fruits results in increased carotenoids, which may have biotechnological potential. PMID:15469492

  9. Enhanced generation of myeloid lineages in hematopoietic differentiation from embryonic stem cells by silencing transcriptional repressor Twist-2.

    PubMed

    Sharabi, Andrew B; Lee, Sung-Hyung; Goodell, Margaret A; Huang, Xue F; Chen, Si-Yi

    2009-12-01

    The self-renewal and multilineage differentiation of embryonic stem cells (ESC) is largely governed by transcription factors or repressors. Extensive efforts have focused on elucidating critical factors that control the differentiation of specific cell lineages, for instance, myeloid lineages in hematopoietic development. In this study, we found that Twist-2, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in inhibiting the differentiation of ESC. Murine ES cells, in which Twist-2 expression is silenced by lentivirally delivered shRNA, exhibit an enhanced formation of primary embryoid bodies (EB) and enhanced differentiation into mesodermally derived hematopoietic colonies. Furthermore, Twist-2 silenced (LV-siTwist-2) ESC display significantly increased generation of myeloid lineages (Gr-1(+) and F4/80(+) cells) during in vitro hematopoietic differentiation. Treatment with the Toll-like receptor (TLR) 4 ligand synergistically stimulates the generation of primary EB formation as well as of hematopoietic progenitors differentiated from LV-siTwist-2 ES cells. Thus, this study reveals the critical role of the transcriptional repressor Twist-2 in regulating the development of myeloid lineage in hematopoietic differentiation from ESC. This study also suggests a potential strategy for directional differentiation of ESC by inhibiting a transcriptional repressor.

  10. Silencing of the PiAvr3a effector-encoding gene from Phytophthora infestans by transcriptional fusion to a short interspersed element.

    PubMed

    Vetukuri, Ramesh R; Tian, Zhendong; Avrova, Anna O; Savenkov, Eugene I; Dixelius, Christina; Whisson, Stephen C

    2011-12-01

    Phytophthora infestans is the notorious oomycete causing late blight of potato and tomato. A large proportion of the P. infestans genome is composed of transposable elements, the activity of which may be controlled by RNA silencing. Accumulation of small RNAs is one of the hallmarks of RNA silencing. Here we demonstrate the presence of small RNAs corresponding to the sequence of a short interspersed retrotransposable element (SINE) suggesting that small RNAs might be involved in silencing of SINEs in P. infestans. This notion was exploited to develop novel tools for gene silencing in P. infestans by engineering transcriptional fusions of the PiAvr3a gene, encoding an RXLR avirulence effector, to the infSINEm retroelement. Transgenic P. infestans lines expressing either 5'-infSINEm::PiAvr3a-3' or 5'-PiAvr3a::SINEm-3' chimeric transcripts initially exhibited partial silencing of PiAvr3a. Over time, PiAvr3a either recovered wild type transcript levels in some lines, or became fully silenced in others. Introduction of an inverted repeat construct was also successful in yielding P. infestans transgenic lines silenced for PiAvr3a. In contrast, constructs expressing antisense or aberrant RNA transcripts failed to initiate silencing of PiAvr3a. Lines exhibiting the most effective silencing of PiAvr3a were either weakly or non-pathogenic on susceptible potato cv. Bintje. This study expands the repertoire of reverse genetics tools available for P. infestans research, and provides insights into a possible mode of variation in effector expression through spread of silencing from adjacent retroelements. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  11. Nucleus-localized antisense small RNAs with 5'-polyphosphate termini regulate long term transcriptional gene silencing in Entamoeba histolytica G3 strain.

    PubMed

    Zhang, Hanbang; Alramini, Hussein; Tran, Vy; Singh, Upinder

    2011-12-30

    In the deep-branching eukaryotic parasite Entamoeba histolytica, transcriptional gene silencing (TGS) of the Amoebapore A gene (ap-a) in the G3 strain has been reported with subsequent development of this parasite strain for gene silencing. However, the mechanisms underlying this gene silencing approach are poorly understood. Here we report that antisense small RNAs (sRNAs) specific to the silenced ap-a gene can be identified in G3 parasites. Furthermore, when additional genes are silenced in the G3 strain, antisense sRNAs to the newly silenced genes can also be detected. Characterization of these sRNAs demonstrates that they are ~27 nucleotides in size, have 5'-polyphosphate termini, and persist even after removal of the silencing plasmid. Immunofluorescence analysis (IFA) and fluorescence in situ hybridization (FISH) show that both the Argonaute protein EhAGO2-2 and antisense sRNAs to the silenced genes are localized to the parasite nucleus. Furthermore, α-EhAGO2-2 immunoprecipitation confirmed the direct association of the antisense sRNAs with EhAGO2-2. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that the loci of the silenced genes are enriched for histone H3 and EhAGO2-2, indicating that both chromatin modification and the RNA-induced transcriptional silencing complex are involved in permanent gene silencing in G3 parasites. In conclusion, our data demonstrate that G3-based gene silencing in E. histolytica is mediated by an siRNA pathway, which utilizes antisense 5'-polyphosphate sRNAs. To our knowledge, this is the first study to show that 5'- polyphosphate antisense sRNAs can mediate TGS, and it is the first example of RNAi-mediated TGS in protozoan parasites.

  12. Media coverage of climate change in Russia: governmental bias and climate silence.

    PubMed

    Poberezhskaya, Marianna

    2015-01-01

    This paper explores which actors and factors influence media coverage of climate change in Russia. It does this by analysing the coverage of three events by five Russian national newspapers (Komsomol'skaya pravda, Rossiyskaya gazeta, Izvestiya, Kommersant and Sovetskaya Rossiya). The three events are the Kyoto Conference in 1997, the Copenhagen Conference in 2009 and the Russian heat-wave of 2010. This paper concludes that regardless of the ownership structure of the newspapers or their dependence on advertising, there is little difference in quantity and quality of overall coverage on climate change. With most newspapers relying on Russian officials as information sources, almost none criticise or question Russian climate policy. Furthermore, the article concludes that, in Russia, the omission of climate change issues from discussion in national newspapers becomes a greater problem than biased coverage, as the lack of commentary decidedly prevents these issues from entering the public debate. © The Author(s) 2014.

  13. Nucleolar Translocation of Histone Deacetylase 2 Is Involved in Regulation of Transcriptional Silencing in the Cat Germinal Vesicle.

    PubMed

    Lee, Pei-Chih; Wildt, David E; Comizzoli, Pierre

    2015-08-01

    Histone deacetylase 2 (HDAC2) is a key transcriptional coregulator that is suspected to play a role during oogenesis. It is known that RNA transcription in the cat germinal vesicle (GV) stops during folliculogenesis at the late antral follicle stage and is unrelated to histone deacetylation or chromatin condensation. The objective of the present study was to determine if and how HDAC2 participates in transcription regulation in the cat GV. Spatiotemporal HDAC2 protein expression was examined by immunostaining oocytes from primary to large antral follicles. HDAC2 was detected in the majority of GVs within oocytes from early, small, and large antral follicles. At early and small antral stages, HDAC2 was found primarily in the GV's nucleoplasm. There then was a significant shift in HDAC2 localization into the nucleolus, mostly in oocytes from large antral follicles. Assessments revealed that transcription was active in oocytes that contained nucleoplasm-localized HDAC2, whereas nucleolar-bound HDAC2 was associated with loss of both global transcription and ribosomal RNA presence at all antral stages. When oocytes were exposed to the HDAC inhibitor valproic acid, results indicated that HDAC regulated transcriptional activity in the nucleoplasm, but not in the nucleolus. Collective results suggest that nucleolar translocation of HDAC2 is associated with transcriptional silencing in the GV, thereby likely contributing to an oocyte's acquisition of competence.

  14. RE1 silencing transcription factor/neuron-restrictive silencing factor regulates expansion of adult mouse subventricular zone-derived neural stem/progenitor cells in vitro.

    PubMed

    Soldati, Chiara; Caramanica, Pasquale; Burney, Matthew J; Toselli, Camilla; Bithell, Angela; Augusti-Tocco, Gabriella; Stanton, Lawrence W; Biagioni, Stefano; Buckley, Noel J; Cacci, Emanuele

    2015-08-01

    Adult neural stem cell (aNSC) activity is tuned by external stimuli through the recruitment of transcription factors. This study examines the RE1 silencing transcription factor (REST) in neural stem/progenitor cells isolated from the subventricular zone of adult mouse brain and provides the first extensive characterization of REST-mediated control of the cellular and molecular properties. This study shows that REST knockdown affects the capacity of progenitor cells to generate neurospheres, reduces cell proliferation, and triggers cell differentiation despite the presence of growth factors. Genome- and transcriptome-wide analyses show that REST binding sites are significantly enriched in genes associated with synaptic transmission and nervous system development and function. Seeking candidate regulators of aNSC function, this study identifies a member of the bone morphogenetic protein (BMP) family, BMP6, the mRNA and protein of which increased after REST knockdown. The results of this study extend previous findings, demonstrating a reciprocal control of REST expression by BMPs. Administration of exogenous BMP6 inhibits aNSC proliferation and induces the expression of the astrocytic marker glial fibrillary acidic protein, highlighting its antimitogenic and prodifferentiative effects. This study suggests that BMP6 produced in a REST-regulated manner together with other signals can contribute to regulation of NSC maintenance and fate.

  15. Salmon: fast and bias-aware quantification of transcript expression using dual-phase inference

    PubMed Central

    Patro, Rob; Duggal, Geet; Love, Michael I; Irizarry, Rafael A; Kingsford, Carl

    2017-01-01

    We introduce Salmon, a method for quantifying transcript abundance from RNA-seq reads that is accurate and fast. Salmon is the first transcriptome-wide quantifier to correct for fragment GC content bias, which we demonstrate substantially improves the accuracy of abundance estimates and the reliability of subsequent differential expression analysis. Salmon combines a new dual-phase parallel inference algorithm and feature-rich bias models with an ultra-fast read mapping procedure. PMID:28263959

  16. Rapid, Cell-Based Toxicity Screen of Potentially Therapeutic Post-Transcriptional Gene Silencing Agents

    PubMed Central

    Kolniak, Tiffany A.; Sullivan, Jack M.

    2011-01-01

    Post-transcriptional gene silencing (PTGS) agents such as antisense, ribozymes and RNA interference (RNAi) have great potential as therapeutics for a variety of eye diseases including retinal and macular degenerations, glaucoma, corneal degenerations, inflammatory and viral conditions. Despite their great potential and over thirty years of academic and corporate research only a single PTGS agent is currently approved for human therapy for a single disease. Substantial challenges exist to achieving both efficacious and safe PTGS agents. Efficacy, as measured in specific target mRNA and protein knockdown, depends upon a number of complex factors including the identification of rare regions of target mRNA accessibility, cellular colocalization of the PTGS agent in sufficient concentration with the target mRNA, and stability of the PTGS agent in the target cells in which it is delivered or expressed. Safety is commonly measured by lack of cytotoxicity or other deleterious cellular responses in cells in which the PTGS agent is delivered or expressed. To relieve major bottlenecks in RNA drug discovery novel, efficient, inexpensive, and rapid tools are needed to facilitate lead identification of the most efficacious PTGS agent, rational optimization of efficacy of the lead agent, and lead agent safety determinations. We have developed a technological platform using cell culture expression systems that permits lead identification and efficacy optimization of PTGS agents against arbitrary disease target mRNAs under relatively high throughput conditions. Here, we extend the technology platform to include PTGS safety determinations in cultured human cells that are expected to represent the common cellular housekeeping microenvironment. We developed a high throughput screening (HTS) cytotoxicity assay in 96-well plate format based around the SYTOX Green dye which is excluded from healthy viable cells and becomes substantially fluorescent only after entering cells and binding

  17. SOX30, a novel epigenetic silenced tumor suppressor, promotes tumor cell apoptosis by transcriptional activating p53 in lung cancer

    PubMed Central

    Han, F; Liu, W; Jiang, X; Shi, X; Yin, L; Ao, L; Cui, Z; Li, Y; Huang, C; Cao, J; Liu, J

    2015-01-01

    Although members of SOX family have been well documented for their essential roles in embryonic development, cell proliferation and disease, the functional role and molecular mechanism of SOX30 in cancer are largely unexplored. Here, we first identified SRY-box containing gene 30 (SOX30) as a novel preferentially methylated gene using genome-wide methylation screening. SOX30 hypermethylation was detected in 100% of lung cancer cell lines (9/9) and 70.83% (85/120) of primary lung tumor tissues compared with none (0/20) of normal and 8.0% (2/25) of peri-tumoral lung tissues (P<0.01). SOX30 was expressed in normal and peri-tumoral lung tissues in which SOX30 was unmethylated, but was silenced or downregulated in lung cancer cell lines and primary lung tumor tissues harboring a hypermethylated SOX30. De-methylation experiments further confirmed that silence of SOX30 was regulated by its hypermethylation. Ectopic expression of SOX30 induces cancer cell apoptosis with inhibiting proliferation in vitro and represses tumor formation in vivo, whereas knockdown of SOX30 demonstrates a reversed effect both in vitro and in vivo. At the molecular level, the antitumorigenic effect of SOX30 is mediated by directly binding to CACTTTG (+115 to +121) of p53 promoter region and activating p53 transcription, suggesting that SOX30 is a novel transcriptional activating factor of p53. Indeed, blockade of p53 attenuates the tumor inhibition of SOX30. Overall, these findings demonstrate that SOX30 is a novel epigenetic silenced tumor suppressor acting through direct regulation of p53 transcription and expression. This study provides novel insights on the mechanism of tumorigenesis in lung cancer. PMID:25435374

  18. Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression.

    PubMed

    Sienski, Grzegorz; Dönertas, Derya; Brennecke, Julius

    2012-11-21

    Eukaryotic genomes are colonized by transposons whose uncontrolled activity causes genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. Here, we show that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila. Genome-wide assays revealed highly correlated changes in RNA polymerase II recruitment, nascent RNA output, and steady-state RNA levels of transposons upon loss of Piwi or Maelstrom. Our data demonstrate piRNA-mediated trans-silencing of hundreds of transposon copies at the transcriptional level. We show that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings. In contrast, loss of Maelstrom affects transposon H3K9me3 patterns only mildly yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work illustrates the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression.

  19. Non-coding transcripts in the H19 imprinting control region mediate gene silencing in transgenic Drosophila.

    PubMed

    Schoenfelder, Stefan; Smits, Guillaume; Fraser, Peter; Reik, Wolf; Paro, Renato

    2007-11-01

    The imprinting control region (ICR) upstream of H19 is the key regulatory element conferring monoallelic expression on H19 and Igf2 (insulin-like growth factor 2). Epigenetic marks in the ICR regulate its interaction with the chromatin protein CCCTC-binding factor and with other control factors to coordinate gene silencing in the imprinting cluster. Here, we show that the H19 ICR is biallelically transcribed, producing both sense and antisense RNAs. We analyse the function of the non-coding transcripts in a Drosophila transgenic system in which the H19 upstream region silences the expression of a reporter gene. We show that knockdown of H19 ICR non-coding RNA (ncRNA) by RNA interference leads to the loss of reporter gene silencing. Our results are, to the best of our knowledge, the first to show that ncRNAs in the H19 ICR are functionally significant, and also indicate that they have a role in regulating gene expression and perhaps epigenetic marks at the H19/Igf2 locus.

  20. Transcriptional Silencing of Transposons by Piwi and Maelstrom and Its Impact on Chromatin State and Gene Expression

    PubMed Central

    Sienski, Grzegorz; Dönertas, Derya; Brennecke, Julius

    2012-01-01

    Summary Eukaryotic genomes are colonized by transposons whose uncontrolled activity causes genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. Here, we show that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila. Genome-wide assays revealed highly correlated changes in RNA polymerase II recruitment, nascent RNA output, and steady-state RNA levels of transposons upon loss of Piwi or Maelstrom. Our data demonstrate piRNA-mediated trans-silencing of hundreds of transposon copies at the transcriptional level. We show that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings. In contrast, loss of Maelstrom affects transposon H3K9me3 patterns only mildly yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work illustrates the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression. PMID:23159368

  1. Switching of dominant retrotransposon silencing strategies from posttranscriptional to transcriptional mechanisms during male germ-cell development in mice

    PubMed Central

    Inoue, Kota; Fukuda, Kei; Sasaki, Hiroyuki

    2017-01-01

    Mammalian genomes harbor millions of retrotransposon copies, some of which are transpositionally active. In mouse prospermatogonia, PIWI-interacting small RNAs (piRNAs) combat retrotransposon activity to maintain the genomic integrity. The piRNA system destroys retrotransposon-derived RNAs and guides de novo DNA methylation at some retrotransposon promoters. However, it remains unclear whether DNA methylation contributes to retrotransposon silencing in prospermatogonia. We have performed comprehensive studies of DNA methylation and polyA(+) RNAs (transcriptome) in developing male germ cells from Pld6/Mitopld and Dnmt3l knockout mice, which are defective in piRNA biogenesis and de novo DNA methylation, respectively. The Dnmt3l mutation greatly reduced DNA methylation levels at most retrotransposons, but its impact on their RNA abundance was limited in prospermatogonia. In Pld6 mutant germ cells, although only a few retrotransposons exhibited reduced DNA methylation, many showed increased expression at the RNA level. More detailed analysis of RNA sequencing, nascent RNA quantification, profiling of cleaved RNA ends, and the results obtained from double knockout mice suggest that PLD6 works mainly at the posttranscriptional level. The increase in retrotransposon expression was larger in Pld6 mutants than it was in Dnmt3l mutants, suggesting that RNA degradation by the piRNA system plays a more important role than does DNA methylation in prospermatogonia. However, DNA methylation had a long-term effect: hypomethylation caused by the Pld6 or Dnmt3l mutation resulted in increased retrotransposon expression in meiotic spermatocytes. Thus, posttranscriptional silencing plays an important role in the early stage of germ cell development, then transcriptional silencing becomes important in later stages. In addition, intergenic and intronic retrotransposon sequences, in particular those containing the antisense L1 promoters, drove ectopic expression of nearby genes in both

  2. The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription.

    PubMed

    Akay, Alper; Di Domenico, Tomas; Suen, Kin M; Nabih, Amena; Parada, Guillermo E; Larance, Mark; Medhi, Ragini; Berkyurek, Ahmet C; Zhang, Xinlian; Wedeles, Christopher J; Rudolph, Konrad L M; Engelhardt, Jan; Hemberg, Martin; Ma, Ping; Lamond, Angus I; Claycomb, Julie M; Miska, Eric A

    2017-08-07

    Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Consolidation of the cancer genome into domains of repressive chromatin by long range epigenetic silencing (LRES) reduces transcriptional plasticity

    PubMed Central

    Coolen, Marcel W.; Stirzaker, Clare; Song, Jenny Z.; Statham, Aaron L.; Kassir, Zena; Moreno, Carlos S.; Young, Andrew N.; Varma, Vijay; Speed, Terence P.; Cowley, Mark; Lacaze, Paul; Kaplan, Warren; Robinson, Mark D.; Clark, Susan J.

    2011-01-01

    SUMMARY Silencing of individual genes can occur by genetic and epigenetic processes during carcinogenesis, but the underlying mechanisms remain unclear. By creating an integrated prostate cancer epigenome map using tiling arrays, we show that contiguous regions of gene suppression commonly occur due to Long Range Epigenetic Silencing (LRES). We identified 47 novel LRES regions in prostate cancer, typically spanning ~2 Mb and harbouring ~12 genes, with a prevalence of tumour suppressor genes and miRNAs. Our data reveal that LRES is associated with regional histone deacetylation combined with sub-domains of different epigenetic remodelling patterns, that include re-enforcement, gain or exchange of repressive histone and DNA methylation marks. The transcriptional and epigenetic state of genes in normal prostate epithelial and human embryonic stem cells can play a critical role in defining the mode of cancer-associated epigenetic remodelling. We propose that a consolidation or effective reduction of the cancer genome commonly occurs in domains, due to a combination of LRES and LOH or genomic deletion, resulting in reduced transcriptional plasticity within these regions. PMID:20173741

  4. Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants

    PubMed Central

    Moreno, Ana Beatriz; Martínez de Alba, Angel Emilio; Bardou, Florian; Crespi, Martin D.; Vaucheret, Hervé; Maizel, Alexis; Mallory, Allison C.

    2013-01-01

    Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5′–3′ exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat–PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm: siRNA-bodies and processing-bodies. We propose a model of single-stranded RNA tug-of-war between RQC and S-PTGS that ensures the correct partitioning of RNA substrates among these RNA degradation pathways. PMID:23482394

  5. Tissue-specific regulation of the rabbit 15-lipoxygenase gene in erythroid cells by a transcriptional silencer.

    PubMed Central

    O'Prey, J; Harrison, P R

    1995-01-01

    The 15-lipoxygenase (lox) gene is expressed in a tissue-specific manner, predominantly in erythroid cells but also in airway epithelial cells and eosinophils. We demonstrate in this report that the 5' flanking DNA of the 15-lox gene contains sequences which down-regulate its activity in a variety of non-erythroid cell lines but not in two erythroid cell lines. The element has characteristics of a transcriptional 'silencer' since it functions in both orientations. The main activity of the silencer has been mapped to the first 900 bp of 5' flanking DNA, which contains nine binding sites for a nuclear factor present in non-erythroid cells but not in erythroid cells. These binding sites have similar sequences and multiple copies of the binding sites confer tissue-specific down-regulation when attached to a minimal lox promoter fragment. The 5' flanking DNA also contains a cluster of three binding sites for the GATA family of transcription factors. Images PMID:7478994

  6. Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants.

    PubMed

    Moreno, Ana Beatriz; Martínez de Alba, Angel Emilio; Bardou, Florian; Crespi, Martin D; Vaucheret, Hervé; Maizel, Alexis; Mallory, Allison C

    2013-04-01

    Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5'-3' exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat-PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm: siRNA-bodies and processing-bodies. We propose a model of single-stranded RNA tug-of-war between RQC and S-PTGS that ensures the correct partitioning of RNA substrates among these RNA degradation pathways.

  7. Self-Serving Biases in Perceiving the Opinions of Others: Implications for the Spiral of Silence (Review Essay).

    ERIC Educational Resources Information Center

    Kennamer, J. David

    1990-01-01

    Synthesizes research supporting the assertion that people are not very accurate perceivers of the opinions of others. Assesses the impact of these findings on E. Noelle-Neumann's "spiral of silence" theory. Suggests an alternative research strategy drawn from social psychology to test this aspect of the spiral of silence process. (SG)

  8. Self-Serving Biases in Perceiving the Opinions of Others: Implications for the Spiral of Silence (Review Essay).

    ERIC Educational Resources Information Center

    Kennamer, J. David

    1990-01-01

    Synthesizes research supporting the assertion that people are not very accurate perceivers of the opinions of others. Assesses the impact of these findings on E. Noelle-Neumann's "spiral of silence" theory. Suggests an alternative research strategy drawn from social psychology to test this aspect of the spiral of silence process. (SG)

  9. REP3-mediated silencing in Saccharomyces cerevisiae.

    PubMed Central

    Papacs, Laurie Ann; Sun, Yu; Anderson, Erica L; Sun, Jianjun; Holmes, Scott G

    2004-01-01

    In yeast the Sir proteins and Rap1p are key regulators of transcriptional silencing at telomeres and the silent mating-type loci. Rap1 and Sir4 also possess anchoring activity; the rotation of plasmids bound by Sir4 or Rap1 is constrained in vivo, and Rap1 or Sir4 binding can also correct the segregation bias of plasmids lacking centromeres. To investigate the mechanistic link between DNA anchoring and regulation of transcription, we examined the ability of a third defined anchor in yeast, the 2micro circle REP3 segregation element, to mediate transcriptional silencing. We find that placement of the REP3 sequence adjacent to the HML locus in a strain deleted for natural silencer sequences confers transcriptional repression on HML. This repression requires the Sir proteins and is decreased in strains lacking the REP3-binding factors Rep1 and Rep2. The yeast cohesin complex associates with REP3; we show that REP3 silencing is also decreased in strains bearing a mutated allele of the MCD1/SCC1 cohesin gene. Conventional silencing is increased in some strains lacking the 2micro circle and decreased in strains overexpressing the Rep1 and Rep2 proteins, suggesting that the Rep proteins antagonize conventional silencing. PMID:15020408

  10. Histone deacetylase homologs regulate epigenetic inheritance of transcriptional silencing and chromosome segregation in fission yeast.

    PubMed

    Grewal, S I; Bonaduce, M J; Klar, A J

    1998-10-01

    Position-effect control at the silent mat2-mat3 interval and at centromeres and telomeres in fission yeast is suggested to be mediated through the assembly of heterochromatin-like structures. Therefore, trans-acting genes that affect silencing may encode either chromatin proteins, factors that modify them, or factors that affect chromatin assembly. Here, we report the identification of an essential gene, clr6 (cryptic loci regulator), which encodes a putative histone deacetylase that when mutated affects epigenetically maintained repression at the mat2-mat3 region and at centromeres and reduces the fidelity of chromosome segregation. Furthermore, we show that the Clr3 protein, when mutated, alleviates recombination block at mat region as well as silencing at donor loci and at centromeres and telomeres, also shares strong homology to known histone deacetylases. Genetic analyses indicate that silencing might be regulated by at least two overlapping histone deacetylase activities. We also found that transient inhibition of histone deacetylase activity by trichostatin A results in the increased missegregation of chromosomes in subsequent generations and, remarkably, alters the imprint at the mat locus, causing the heritable conversion of the repressed epigenetic state to the expressed state. This work supports the model that the level of histone deacetylation has a role in the assembly of repressive heterochromatin and provides insight into the mechanism of epigenetic inheritance.

  11. Transcriptional silencing and activation of paternal DNA during P lasmodium berghei zygotic development and transformation to oocyst

    PubMed Central

    Ukegbu, Chiamaka V.; Cho, Jee‐Sun; Christophides, George K.

    2015-01-01

    Summary The malaria parasite develops sexually in the mosquito midgut upon entry with the ingested blood meal before it can invade the midgut epithelium and embark on sporogony. Recent data have identified a number of distinct transcriptional programmes operating during this critical phase of the parasite life cycle. We aimed at characterizing the parental contribution to these transcriptional programmes and establish the genetic framework that would guide further studies of P lasmodium zygotic development and ookinete‐to‐oocyst transition. To achieve this we used in vitro and in vivo cross‐fertilization experiments of various parasite lines expressing fluorescent reporters under the control of constitutive and stage‐specific promoters. The results revealed that the zygote/ookinete stage exhibits a maternal phenotype with respect to constitutively expressed reporters, which is derived from either maternal mRNA inheritance or transcription of the maternal allele. The respective paternal alleles are silenced in the zygote/ookinete but reactivated after midgut invasion and transformation to oocyst. Transcripts specifically produced in the zygote/ookinete are synthesized de novo by both parental alleles. These findings highlight a putative role of epigenetic regulation of P lasmodium zygotic development and add substantially to the emerging picture of the molecular mechanisms regulating this important stage of malaria transmission. PMID:25728487

  12. The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals

    PubMed Central

    Wiegand, Stephan; Meier, Doreen; Seehafer, Carsten; Malicki, Marek; Hofmann, Patrick; Schmith, Anika; Winckler, Thomas; Földesi, Balint; Boesler, Benjamin; Nellen, Wolfgang; Reimegård, Johan; Käller, Max; Hällman, Jimmie; Emanuelsson, Olof; Avesson, Lotta; Söderbom, Fredrik; Hammann, Christian

    2014-01-01

    Dictyostelium intermediate repeat sequence 1 (DIRS-1) is the founding member of a poorly characterized class of retrotransposable elements that contain inverse long terminal repeats and tyrosine recombinase instead of DDE-type integrase enzymes. In Dictyostelium discoideum, DIRS-1 forms clusters that adopt the function of centromeres, rendering tight retrotransposition control critical to maintaining chromosome integrity. We report that in deletion strains of the RNA-dependent RNA polymerase RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. Shorter versions of a hitherto unknown long non-coding RNA in DIRS-1 antisense orientation are also enriched in rrpC– strains. Concurrent with the accumulation of long transcripts, the vast majority of small (21 mer) DIRS-1 RNAs vanish in rrpC– strains. RNASeq reveals an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation. We show that RrpC is required for post-transcriptional DIRS-1 silencing and also for spreading of RNA silencing signals. Finally, DIRS-1 mis-regulation in the absence of RrpC leads to retrotransposon mobilization. In summary, our data reveal RrpC as a key player in the silencing of centromeric retrotransposon DIRS-1. RrpC acts at the post-transcriptional level and is involved in spreading of RNA silencing signals, both in the 5′ and 3′ directions. PMID:24369430

  13. Strong spurious transcription likely contributes to DNA insert bias in typical metagenomic clone libraries.

    PubMed

    Lam, Kathy N; Charles, Trevor C

    2015-01-01

    Clone libraries provide researchers with a powerful resource to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, and allowed the mining of novel enzymes. Libraries are often constructed by cloning large inserts into cosmid or fosmid vectors. Recently, there have been reports of GC bias in fosmid metagenomic libraries, and it was speculated to be a result of fragmentation and loss of AT-rich sequences during cloning. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role. To explore possible mechanisms responsible for sequence bias in clone libraries, we constructed a cosmid library from a human microbiome sample and sequenced DNA from different steps during library construction: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final cosmid library, and we provide evidence that the bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after DNA is introduced into Escherichia coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for promoters and found that rpoD/σ(70) promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found the bias to be more correlated with the number of rpoD/σ(70) consensus sequences in the genome than with simple GC content. The GC bias of metagenomic libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/σ(70) consensus-like in E. coli may lead to instability, causing loss of the plasmid or loss of the insert DNA that gives rise to the transcription. Despite

  14. Transcriptional Gene Silencing Maintained by OTS1 SUMO Protease Requires a DNA-Dependent Polymerase V-Dependent Pathway1[OPEN

    PubMed Central

    Liu, Lei; Yan, Xiaojing; Zhao, Yiqiang

    2017-01-01

    The expression of genes with aberrant structure is prevented at both the transcriptional and posttranscriptional regulation levels. Aberrant gene silencing at the posttranscriptional level is well studied; however, it is not well understood how aberrant genes are silenced at the transcriptional level. In this study, through genetic screening a transgenic report line that harbors an aberrant gene (35S-LUC, lacking 3′-untranslated region [3′-UTR]) and lacks luciferase (LUC) activity, we identify that the small ubiquitin-like modifier (SUMO) protease OTS1 gene is required for maintaining the silence of the reporter 35S-LUC and an endogenous mutator-like element MULE-F19G14 at the transcriptional level, which requires DNA-dependent RNA polymerase (Pol) V and DDR complex, but not Pol IV. The increased transcripts in ots1 mutants are terminated by the 3′-UTRs of downstream genes. In addition to ots1 mutations, mutations in several known or putative SUMO proteases and two SUMO E3 ligases, SIZ1 and MMS21, have similar effects on this silencing regulation. Taken together, our results reveal that the enzymes involved in the SUMOylation process restrain aberrant gene transcription by using a downstream gene 3′-UTR, and this regulation requires a functional Pol V-dependent pathway in Arabidopsis (Arabidopsis thaliana). PMID:27852949

  15. Silencing clusterin gene transcription on effects of multidrug resistance reversing of human hepatoma HepG2/ADM cells.

    PubMed

    Zheng, Wenjie; Sai, Wenli; Yao, Min; Gu, Hongbin; Yao, Yao; Qian, Qi; Yao, Dengfu

    2015-05-01

    Abnormal clusterin (CLU) expression is associated with multidrug resistance (MDR) of hepatocellular carcinoma (HCC). In the present study, the CLU expression was analyzed in human hepatoma cells and chemoresistant counterpart HepG2/ADM cells. Compared with L02 cells, the overexpression of cellular CLU was identified in HepG2, HepG2/ADM, SMMC7721, Hep3B ,and PLC cells and relatively lower expression in Bel-7404, SNU-739, and MHCC97H cells. Specific short hairpin RNAs (shRNAs) to silence CLU gene transcription were designed, and the most effective sequences were screened. After the HepG2/ADM cells transfected with shRNA-1, the inhibition of CLU expression was 73.68 % at messenger RNA (mRNA) level by real-time quantitative RT-PCR with obvious enhancement in cell chemosensitivity, increasing apoptosis induced by doxorubicin using fluorescence kit, and Rh-123 retention qualified with flow cytometry. Knockdown CLU also significantly decreased the drug efflux pump activity through the depression of MDR1/P-glycoprotein (q = 11.739, P < 0.001). Moreover, silencing CLU led to downregulation of β-catenin (q = 13.544, P = 0.001), suggesting that downregulation of CLU might be a key point to reverse multidrug resistance of HepG2/ADM cells.

  16. White as a Reporter Gene to Detect Transcriptional Silencers Specifying Position-Specific Gene Expression during Drosophila Melanogaster Eye Development

    PubMed Central

    Sun, Y. H.; Tsai, C. J.; Green, M. M.; Chao, J. L.; Yu, C. T.; Jaw, T. J.; Yeh, J. Y.; Bolshakov, V. N.

    1995-01-01

    The white(+) gene was used as a reporter to detect transcriptional silencer activity in the Drosophila genome. Changes in the spatial expression pattern of white were scored in the adult eye as nonuniform patterns of pigmentation. Thirty-six independent P[lacW] transposant lines were collected. These represent 12 distinct pigmentation patterns and probably 21 loci. The spatial pigmentation pattern is due to cis-acting suppression of white(+) expression, and the suppression probably depends on cell position rather than cell type. The mechanism of suppression differs from inactivation by heterochromatin. In addition, activation of lacZ in P[lacW] occurs also in specific patterns in imaginal discs and embryos in many of the lines. The expression patterns of white(+) and lacZ may reflect the activity of regulatory elements belonging to an endogenous gene near each P[lacW] insertion site. We speculate that these putative POSE (position-specific expression) genes may have a role in pattern formation of the eye as well as other imaginal structures. Three of the loci identified are optomotor-blind, engrailed and invected. teashirt is also implicated as a candidate gene. We propose that this ``silencer trap'' may be an efficient way of identifying genes involved in imaginal pattern formation. PMID:8582614

  17. 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.

  18. The post-transcriptional gene silencing machinery functions independently of DNA methylation to repress a LINE1-like retrotransposon in Neurospora crassa

    PubMed Central

    Nolan, Tony; Braccini, Laura; Azzalin, Gianluca; De Toni, Arianna; Macino, Giuseppe; Cogoni, Carlo

    2005-01-01

    Post-transcriptional gene silencing (PTGS) involving small interfering RNA (siRNA)-directed degradation of RNA transcripts and transcriptional silencing via DNA methylation have each been proposed as mechanisms of genome defence against invading nucleic acids, such as transposons and viruses. Furthermore, recent data from plants indicates that many transposons are silenced via a combination of the two mechanisms, and siRNAs can direct methylation of transposon sequences. We investigated the contribution of DNA methylation and the PTGS pathway to transposon control in the filamentous fungus Neurospora crassa. We found that repression of the LINE1-like transposon, Tad, requires the Argonaute protein QDE2 and Dicer, each of which are required for transgene-induced PTGS (quelling) in N.crassa. Interestingly, unlike quelling, the RNA-dependent RNA polymerase QDE1 and the RecQ DNA helicase QDE3 were not required for Tad control, suggesting the existence of specialized silencing pathways for diverse kinds of repetitive elements. In contrast, Tad elements were not significantly methylated and the DIM2 DNA methyltransferase, responsible for all known DNA methylation in Neurospora, had no effect on Tad control. Thus, an RNAi-related transposon silencing mechanism operates during the vegetative phase of N.crassa that is independent of DNA methylation, highlighting a major difference between this organism and other methylation-proficient species. PMID:15767281

  19. Expression profiling, gene silencing and transcriptional networking of metzincin metalloproteases in the cattle tick, Rhipicephalus (Boophilus) microplus.

    PubMed

    Barnard, Annette-Christi; Nijhof, Ard M; Gaspar, Anabella R M; Neitz, Albert W H; Jongejan, Frans; Maritz-Olivier, Christine

    2012-05-25

    Tick proteins functioning in vital physiological processes such as blood meal uptake, digestion and reproduction are potential targets for anti-tick vaccines, since vaccination could inhibit these essential functions and ultimately affect tick survival. In this study we identified metzincin metalloproteases from Rhipicephalus microplus as potential vaccine candidates since they are implicated as essential to blood-cavity formation, bloodmeal digestion and reproduction in ixodid ticks. Eight transcripts encoding proteins that contain the characteristic metzincin zinc-binding motif HEXXHXXG/NXXH/D and a unique methionine containing "methionine-turn" were identified from native and in-house assembled R. microplus expressed sequence tag (EST) databases. These were representative of five reprolysin-like and three astacin-like metzincin metalloproteases. Reverse transcription-PCR analysis indicated that the reprolysins were most abundantly expressed in the salivary glands, whereas the astacins were most abundant in the midgut and ovaries. In vivo gene silencing was performed to assess a possible phenotype of these metalloproteases during adult female R. microplus blood feeding and reproduction. RNA interference (RNAi) against two of the reprolysins and one of the astacins significantly affected the average egg weight and oviposition rate. Evidently, this reverse genetic approach enabled the evaluation of the overall vital impact of tick proteins. Finally, integrated real time-PCR studies also revealed an extensive cross organ network between the R. microplus metzincin transcripts, supporting the use of a combinatorial metzincin-based anti- R. microplus vaccine. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling.

    PubMed

    Bouhouche, Khaled; Gout, Jean-François; Kapusta, Aurélie; Bétermier, Mireille; Meyer, Eric

    2011-05-01

    Proteins of the Argonaute family are small RNA carriers that guide regulatory complexes to their targets. The family comprises two major subclades. Members of the Ago subclade, which are present in most eukaryotic phyla, bind different classes of small RNAs and regulate gene expression at both transcriptional and post-transcriptional levels. Piwi subclade members appear to have been lost in plants and fungi and were mostly studied in metazoa, where they bind piRNAs and have essential roles in sexual reproduction. Their presence in ciliates, unicellular organisms harbouring both germline micronuclei and somatic macronuclei, offers an interesting perspective on the evolution of their functions. Here, we report phylogenetic and functional analyses of the 15 Piwi genes from Paramecium tetraurelia. We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle. Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus. Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements.

  1. Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling

    PubMed Central

    Bouhouche, Khaled; Gout, Jean-François; Kapusta, Aurélie; Bétermier, Mireille; Meyer, Eric

    2011-01-01

    Proteins of the Argonaute family are small RNA carriers that guide regulatory complexes to their targets. The family comprises two major subclades. Members of the Ago subclade, which are present in most eukaryotic phyla, bind different classes of small RNAs and regulate gene expression at both transcriptional and post-transcriptional levels. Piwi subclade members appear to have been lost in plants and fungi and were mostly studied in metazoa, where they bind piRNAs and have essential roles in sexual reproduction. Their presence in ciliates, unicellular organisms harbouring both germline micronuclei and somatic macronuclei, offers an interesting perspective on the evolution of their functions. Here, we report phylogenetic and functional analyses of the 15 Piwi genes from Paramecium tetraurelia. We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle. Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus. Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements. PMID:21216825

  2. MIDGET unravels functions of the Arabidopsis topoisomerase VI complex in DNA endoreduplication, chromatin condensation, and transcriptional silencing.

    PubMed

    Kirik, Viktor; Schrader, Andrea; Uhrig, Joachim F; Hulskamp, Martin

    2007-10-01

    The plant homologs of the archaeal DNA topoisomerase VI complex are required for the progression of endoreduplication cycles. Here, we describe the identification of MIDGET (MID) as a novel component of topoisomerase VI. We show that mid mutants show the same phenotype as rhl1, rhl2, and top6B mutants and that MID protein physically interacts with RHL1. The phenotypic analysis revealed new phenotypes, indicating that topoisomerase VI is involved in chromatin organization and transcriptional silencing. In addition, genetic evidence is provided suggesting that the ATR-dependent DNA damage repair checkpoint is activated in mid mutants, and CYCB1;1 is ectopically activated. Finally, we demonstrate that overexpression of CYCB1;2 can rescue the endoreduplication defects in mid mutants, suggesting that in mid mutants, a specific checkpoint is activated preventing further progression of endoreduplication cycles.

  3. NF-kappaB regulation of YY1 inhibits skeletal myogenesis through transcriptional silencing of myofibrillar genes.

    PubMed

    Wang, Huating; Hertlein, Erin; Bakkar, Nadine; Sun, Hao; Acharyya, Swarnali; Wang, Jingxin; Carathers, Micheal; Davuluri, Ramana; Guttridge, Denis C

    2007-06-01

    NF-kappaB signaling is implicated as an important regulator of skeletal muscle homeostasis, but the mechanisms by which this transcription factor contributes to muscle maturation and turnover remain unclear. To gain insight into these mechanisms, gene expression profiling was examined in C2C12 myoblasts devoid of NF-kappaB activity. Interestingly, even in proliferating myoblasts, the absence of NF-kappaB caused the pronounced induction of several myofibrillar genes, suggesting that NF-kappaB functions as a negative regulator of late-stage muscle differentiation. Although several myofibrillar promoters contain predicted NF-kappaB binding sites, functional analysis using the troponin-I2 gene as a model revealed that NF-kappaB-mediated repression does not occur through direct DNA binding. In the search for an indirect mediator, the transcriptional repressor YinYang1 (YY1) was identified. While inducers of NF-kappaB stimulated YY1 expression in multiple cell types, genetic ablation of the RelA/p65 subunit of NF-kappaB in both cultured cells and adult skeletal muscle correlated with reduced YY1 transcripts and protein. NF-kappaB regulation of YY1 occurred at the transcriptional level, mediated by direct binding of the p50/p65 heterodimer complex to the YY1 promoter. Furthermore, YY1 was found associated with multiple myofibrillar promoters in C2C12 myoblasts containing NF-kappaB activity. Based on these results, we propose that NF-kappaB regulation of YY1 and transcriptional silencing of myofibrillar genes represent a new mechanism by which NF-kappaB functions in myoblasts to modulate skeletal muscle differentiation.

  4. NF-κB Regulation of YY1 Inhibits Skeletal Myogenesis through Transcriptional Silencing of Myofibrillar Genes▿ †

    PubMed Central

    Wang, Huating; Hertlein, Erin; Bakkar, Nadine; Sun, Hao; Acharyya, Swarnali; Wang, Jingxin; Carathers, Micheal; Davuluri, Ramana; Guttridge, Denis C.

    2007-01-01

    NF-κB signaling is implicated as an important regulator of skeletal muscle homeostasis, but the mechanisms by which this transcription factor contributes to muscle maturation and turnover remain unclear. To gain insight into these mechanisms, gene expression profiling was examined in C2C12 myoblasts devoid of NF-κB activity. Interestingly, even in proliferating myoblasts, the absence of NF-κB caused the pronounced induction of several myofibrillar genes, suggesting that NF-κB functions as a negative regulator of late-stage muscle differentiation. Although several myofibrillar promoters contain predicted NF-κB binding sites, functional analysis using the troponin-I2 gene as a model revealed that NF-κB-mediated repression does not occur through direct DNA binding. In the search for an indirect mediator, the transcriptional repressor YinYang1 (YY1) was identified. While inducers of NF-κB stimulated YY1 expression in multiple cell types, genetic ablation of the RelA/p65 subunit of NF-κB in both cultured cells and adult skeletal muscle correlated with reduced YY1 transcripts and protein. NF-κB regulation of YY1 occurred at the transcriptional level, mediated by direct binding of the p50/p65 heterodimer complex to the YY1 promoter. Furthermore, YY1 was found associated with multiple myofibrillar promoters in C2C12 myoblasts containing NF-κB activity. Based on these results, we propose that NF-κB regulation of YY1 and transcriptional silencing of myofibrillar genes represent a new mechanism by which NF-κB functions in myoblasts to modulate skeletal muscle differentiation. PMID:17438126

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

    PubMed

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

    2012-09-01

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

  6. The SUMO E3 Ligase-Like Proteins PIAL1 and PIAL2 Interact with MOM1 and Form a Novel Complex Required for Transcriptional Silencing.

    PubMed

    Han, Yong-Feng; Zhao, Qiu-Yuan; Dang, Liang-Liang; Luo, Yu-Xi; Chen, Shan-Shan; Shao, Chang-Rong; Huang, Huan-Wei; Li, Yong-Qiang; Li, Lin; Cai, Tao; Chen, She; He, Xin-Jian

    2016-05-01

    The mechanism by which MORPHEUS' MOLECULE1 (MOM1) contributes to transcriptional gene silencing has remained elusive since the gene was first identified and characterized. Here, we report that two Arabidopsis thaliana PIAS (PROTEIN INHIBITOR OF ACTIVATED STAT)-type SUMO E3 ligase-like proteins, PIAL1 and PIAL2, function redundantly to mediate transcriptional silencing at MOM1 target loci. PIAL1 and PIAL2 physically interact with each other and with MOM1 to form a high molecular mass complex. In the absence of either PIAL2 or MOM1, the formation of the high molecular mass complex is disrupted. We identified a previously uncharacterized IND (interacting domain) in PIAL1 and PIAL2 and demonstrated that IND directly interacts with MOM1. The CMM2 (conserved MOM1 motif 2) domain of MOM1 was previously shown to be required for the dimerization of MOM1. We demonstrated that the CMM2 domain is also required for the interaction of MOM1 with PIAL1 and PIAL2. We found that although PIAL2 has SUMO E3 ligase activity, the activity is dispensable for PIAL2's function in transcriptional silencing. This study suggests that PIAL1 and PIAl2 act as components of the MOM1-containing complex to mediate transcriptional silencing at heterochromatin regions.

  7. Control of transcriptional pausing by biased thermal fluctuations on repetitive genomic sequences

    PubMed Central

    Imashimizu, Masahiko; Afek, Ariel; Takahashi, Hiroki; Lubkowska, Lucyna; Lukatsky, David B.

    2016-01-01

    In the process of transcription elongation, RNA polymerase (RNAP) pauses at highly nonrandom positions across genomic DNA, broadly regulating transcription; however, molecular mechanisms responsible for the recognition of such pausing positions remain poorly understood. Here, using a combination of statistical mechanical modeling and high-throughput sequencing and biochemical data, we evaluate the effect of thermal fluctuations on the regulation of RNAP pausing. We demonstrate that diffusive backtracking of RNAP, which is biased by repetitive DNA sequence elements, causes transcriptional pausing. This effect stems from the increased microscopic heterogeneity of an elongation complex, and thus is entropy-dominated. This report shows a linkage between repetitive sequence elements encoded in the genome and regulation of RNAP pausing driven by thermal fluctuations. PMID:27830653

  8. Epigenetic silencing of Bim transcription by Spi-1/PU.1 promotes apoptosis resistance in leukaemia

    PubMed Central

    Ridinger-Saison, M; Evanno, E; Gallais, I; Rimmelé, P; Selimoglu-Buet, D; Sapharikas, E; Moreau-Gachelin, F; Guillouf, C

    2013-01-01

    Deregulation of transcriptional networks contributes to haematopoietic malignancies. The transcription factor Spi-1/PU.1 is a master regulator of haematopoiesis and its alteration leads to leukaemia. Spi-1 overexpression inhibits differentiation and promotes resistance to apoptosis in erythroleukaemia. Here, we show that Spi-1 inhibits mitochondrial apoptosis in vitro and in vivo through the transcriptional repression of Bim, a proapoptotic factor. BIM interacts with MCL-1 that behaves as a major player in the survival of the preleukaemic cells. The repression of BIM expression reduces the amount of BIM-MCL-1 complexes, thus increasing the fraction of potentially active antiapoptotic MCL-1. We then demonstrate that Spi-1 represses Bim transcription by binding to the Bim promoter and by promoting the trimethylation of histone 3 on lysine 27 (H3K27me3, a repressive histone mark) on the Bim promoter. The PRC2 repressive complex of Polycomb is directly responsible for the deposit of H3K27me3 mark at the Bim promoter. SUZ12 and the histone methyltransferase EZH2, two PRC2 subunits bind to the Bim promoter at the same location than H3K27me3, distinct of the Spi-1 DNA binding site. As Spi-1 interacts with SUZ12 and EZH2, these results indicate that Spi-1 modulates the activity of PRC2 without directly recruiting the complex to the site of its activity on the chromatin. Our results identify a new mechanism whereby Spi-1 represses transcription and provide mechanistic insights on the antiapoptotic function of a transcription factor mediated by the epigenetic control of gene expression. PMID:23852375

  9. Epigenetic silencing of Bim transcription by Spi-1/PU.1 promotes apoptosis resistance in leukaemia.

    PubMed

    Ridinger-Saison, M; Evanno, E; Gallais, I; Rimmelé, P; Selimoglu-Buet, D; Sapharikas, E; Moreau-Gachelin, F; Guillouf, C

    2013-09-01

    Deregulation of transcriptional networks contributes to haematopoietic malignancies. The transcription factor Spi-1/PU.1 is a master regulator of haematopoiesis and its alteration leads to leukaemia. Spi-1 overexpression inhibits differentiation and promotes resistance to apoptosis in erythroleukaemia. Here, we show that Spi-1 inhibits mitochondrial apoptosis in vitro and in vivo through the transcriptional repression of Bim, a proapoptotic factor. BIM interacts with MCL-1 that behaves as a major player in the survival of the preleukaemic cells. The repression of BIM expression reduces the amount of BIM-MCL-1 complexes, thus increasing the fraction of potentially active antiapoptotic MCL-1. We then demonstrate that Spi-1 represses Bim transcription by binding to the Bim promoter and by promoting the trimethylation of histone 3 on lysine 27 (H3K27me3, a repressive histone mark) on the Bim promoter. The PRC2 repressive complex of Polycomb is directly responsible for the deposit of H3K27me3 mark at the Bim promoter. SUZ12 and the histone methyltransferase EZH2, two PRC2 subunits bind to the Bim promoter at the same location than H3K27me3, distinct of the Spi-1 DNA binding site. As Spi-1 interacts with SUZ12 and EZH2, these results indicate that Spi-1 modulates the activity of PRC2 without directly recruiting the complex to the site of its activity on the chromatin. Our results identify a new mechanism whereby Spi-1 represses transcription and provide mechanistic insights on the antiapoptotic function of a transcription factor mediated by the epigenetic control of gene expression.

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

    PubMed Central

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

    1993-01-01

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

  11. Cbx7 is epigenetically silenced in glioblastoma and inhibits cell migration by targeting YAP/TAZ-dependent transcription

    PubMed Central

    Nawaz, Zahid; Patil, Vikas; Arora, Anjali; Hegde, Alangar S.; Arivazhagan, Arimappamagan; Santosh, Vani; Somasundaram, Kumaravel

    2016-01-01

    Glioblastomas (GBM) are the most malignant form of astrocytomas which are difficult to treat and portend a grave clinical course and poor prognosis. In this study, we identified Chromobox homolog 7 (Cbx7), a member of Polycomb Repressive Complex 1 (PRC1), as a downregulated gene in GBM owing to its promoter hypermethylation. Bisulphite sequencing and methylation inhibitor treatment established the hypermethylation of Cbx7 in GBM. Exogenous overexpression of Cbx7 induced cell death, inhibited cell proliferation, colony formation and migration/invasion of the glioma cells. GSEA of Cbx7 regulated genes identified Cbx7 as a repressor of transcription co-activators YAP/TAZ, the inhibitory targets of the Hippo signalling pathway. In good correlation, the exogenous expression of Cbx7 repressed the YAP/TAZ-dependent transcription and downregulated CTGF, a bonafide YAP/TAZ target. We also observed reduced levels of phospho-JNK in Cbx7 expressing cells. Additionally, CTGF silencing and pharmacological inhibition of JNK also inhibited glioma cell migration. Further, Cbx7 failed to inhibit cell migration significantly in the presence of exogenously overexpressed CTGF or constitutively active JNK. Thus, our study identifies Cbx7 as an inhibitor of glioma cell migration through its inhibitory effect on YAP/TAZ-CTGF-JNK signalling axis and underscores the importance of epigenetic inactivation of Cbx7 in gliomagenesis. PMID:27291091

  12. Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p

    PubMed Central

    Nguyen, Ha T.; Jia, Wei; Beedle, Aaron M.; Kennedy, Eileen J.; Murph, Mandi M.

    2015-01-01

    Although microRNAs (miRNAs) are small, non-protein-coding entities, they have important roles in post-transcriptional regulation of most of the human genome. These small entities generate fine-tuning adjustments in the expression of mRNA, which can mildly or massively affect the abundance of proteins. Previously, we found that the expression of miR-30c-2-3p is induced by lysophosphatidic acid and has an important role in the regulation of cell proliferation in ovarian cancer cells. The goal here is to confirm that ATF3 mRNA is a target of miR-30c-2-3p silencing, thereby further establishing the functional role of miR-30c-2-3p. Using a combination of bioinformatics, qRT-PCR, immunoblotting and luciferase assays, we uncovered a regulatory pathway between miR-30c-2-3p and the expression of the transcription factor, ATF3. Lysophosphatidic acids triggers the expression of both miR-30c-2-3p and ATF3, which peak at 1 h and are absent 8 h post stimulation in SKOV-3 and OVCAR-3 serous ovarian cancer cells. The 3´-untranslated region (3´-UTR) of ATF3 was a predicted, putative target for miR-30c-2-3p, which we confirmed as a bona-fide interaction using a luciferase reporter assay. Specific mutations introduced into the predicted site of interaction between miR-30c-2-3p and the 3´-UTR of ATF3 alleviated the suppression of the luciferase signal. Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after lysophosphatidic acid stimulation. Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by lysophosphatidic acid, subsequently miR-30c-2-3p negatively regulates the expression of ATF3 through post-transcriptional silencing, which prevents further ATF3-related outcomes as a consequence of lysophosphatidic acid signaling. PMID:26418018

  13. Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p.

    PubMed

    Nguyen, Ha T; Jia, Wei; Beedle, Aaron M; Kennedy, Eileen J; Murph, Mandi M

    2015-01-01

    Although microRNAs (miRNAs) are small, non-protein-coding entities, they have important roles in post-transcriptional regulation of most of the human genome. These small entities generate fine-tuning adjustments in the expression of mRNA, which can mildly or massively affect the abundance of proteins. Previously, we found that the expression of miR-30c-2-3p is induced by lysophosphatidic acid and has an important role in the regulation of cell proliferation in ovarian cancer cells. The goal here is to confirm that ATF3 mRNA is a target of miR-30c-2-3p silencing, thereby further establishing the functional role of miR-30c-2-3p. Using a combination of bioinformatics, qRT-PCR, immunoblotting and luciferase assays, we uncovered a regulatory pathway between miR-30c-2-3p and the expression of the transcription factor, ATF3. Lysophosphatidic acids triggers the expression of both miR-30c-2-3p and ATF3, which peak at 1 h and are absent 8 h post stimulation in SKOV-3 and OVCAR-3 serous ovarian cancer cells. The 3´-untranslated region (3´-UTR) of ATF3 was a predicted, putative target for miR-30c-2-3p, which we confirmed as a bona-fide interaction using a luciferase reporter assay. Specific mutations introduced into the predicted site of interaction between miR-30c-2-3p and the 3´-UTR of ATF3 alleviated the suppression of the luciferase signal. Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after lysophosphatidic acid stimulation. Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by lysophosphatidic acid, subsequently miR-30c-2-3p negatively regulates the expression of ATF3 through post-transcriptional silencing, which prevents further ATF3-related outcomes as a consequence of lysophosphatidic acid signaling.

  14. Ume6 Is Required for the MATa/MATα Cellular Identity and Transcriptional Silencing in Kluyveromyces lactis

    PubMed Central

    Barsoum, E.; Sjöstrand, J. O. O.; Åström, S. U.

    2010-01-01

    To explore the similarities and differences of regulatory circuits among budding yeasts, we characterized the role of the unscheduled meiotic gene expression 6 (UME6) gene in Kluyveromyces lactis. We found that Ume6 was required for transcriptional silencing of the cryptic mating-type loci HMLα and HMRa. Chromatin immunoprecipitation (ChIP) suggested that Ume6 acted directly by binding the cis-regulatory silencers of these loci. Unexpectedly, a MATa ume6 strain was mating proficient, whereas a MATα ume6 strain was sterile. This observation was explained by the fact that ume6 derepressed HMLα2 only weakly, but derepressed HMRa1 strongly. Consistently, two a/α-repressed genes (MTS1 and STE4) were repressed in the MATα ume6 strain, but were expressed in the MATa ume6 strain. Surprisingly, ume6 partially suppressed the mating defect of a MATa sir2 strain. MTS1 and STE4 were repressed in the MATa sir2 ume6 double-mutant strain, indicating that the suppression acted downstream of the a1/α2-repressor. We show that both STE12 and the MATa2/HMRa2 genes were overexpressed in the MATa sir2 ume6 strain. Consistent with the idea that this deregulation suppressed the mating defect, ectopic overexpression of Ste12 and a2 in a MATa sir2 strain resulted in efficient mating. In addition, Ume6 served as a block to polyploidy, since ume6/ume6 diploids mated as pseudo a-strains. Finally, Ume6 was required for repression of three meiotic genes, independently of the Rpd3 and Sin3 corepressors. PMID:20139343

  15. Development of a luciferase-based reporter of transcriptional gene silencing that enables bidirectional mutant screening in Arabidopsis thaliana

    PubMed Central

    2012-01-01

    Background Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis. Results We introduced a dual 35 S promoter (d35S) driven luciferase reporter, LUCH, into Arabidopsis and isolated a line with a moderate level of luciferase activity. LUCH produced transgene-specific 24 nucleotide siRNAs and its d35S contained methylated cytosine in CG, CHG and CHH contexts. Treatment of the transgenic line with an inhibitor of cytosine methylation de-repressed luciferase activity. Mutations in several components of the RdDM pathway but not the maintenance methylation genes resulted in reduced d35S methylation, especially CHH methylation, and de-repression of luciferase activity. A mutation in MOM1, which is known to cooperate with RdDM to silence transposons, reduced d35S DNA methylation and de-repressed LUCH expression. A mutation in ROS1, a cytosine demethylation enzyme, increased d35S methylation and reduced LUCH expression. Conclusion We developed a luciferase-based reporter, LUCH, which reports both DNA methylation directed by small RNAs and active demethylation by ROS1 in Arabidopsis. The moderate basal level of LUCH expression allows for bi-directional genetic screens that dissect the mechanisms of DNA methylation as well as demethylation. PMID:22676624

  16. Involvement of argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter

    PubMed Central

    Chu, Yongjun; Yue, Xuan; Younger, Scott T.; Janowski, Bethany A.; Corey, David R.

    2010-01-01

    Double-stranded RNAs that are complementary to non-coding transcripts at gene promoters can activate or inhibit gene expression in mammalian cells. Understanding the mechanism for modulating gene expression by promoter-targeted antigene RNAs (agRNAs) will require identification of the proteins involved in recognition. Previous reports have implicated argonaute (AGO) proteins, but identifications have differed with involvement of AGO1, AGO2, or both AGO1 and AGO2 being reported by different studies. The roles of AGO3 and AGO4 have not been investigated. Here, we examine the role of AGO 1–4 in gene silencing and activation of the progesterone receptor (PR) gene. Expression of AGO2 is necessary for efficient gene silencing or activation and AGO2 is recruited to the non-coding transcript that overlaps the promoter during both gene silencing and activation. Expression of AGO1, AGO3 and AGO4 are not necessary for gene silencing or activation nor are AGO1, AGO3, or AGO4 recruited to the target non-coding transcript during gene activation. These data indicate that AGO2 is the primary AGO variant involved in modulating expression of PR by agRNAs. PMID:20675357

  17. CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

    PubMed

    Yu, Zhiyuan; Kong, Qun; Kone, Bruce C

    2010-03-01

    Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

  18. Three gene products of a begomovirus-betasatellite complex restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana.

    PubMed

    Saeed, Muhammad; Krczal, Gabi; Wassenegger, Michael

    2015-04-01

    Single-stranded DNA geminiviruses replicate via double-stranded DNA intermediates forming mini-chromosomes that are targets for transcriptional gene silencing (TGS) in plants. The ability of the cotton leaf curl Kokhran virus (CLCuKoV)-cotton leaf curl Multan betasatellite (CLCuMuB) proteins, replication-associated protein (Rep), transcriptional activator protein (TrAP), C4, V2 and βC1, to suppress TGS was investigated by using the Nicotiana benthamiana line 16-TGS (16-TGS) harbouring a transcriptionally silenced green fluorescent protein (GFP) transgene. Inoculation of 16-TGS plants with a recombinant potato virus X vector carrying Rep, TrAP or βC1 resulted in re-expression of GFP. Northern blot analysis confirmed that the observed GFP fluorescence was associated with GFP mRNA accumulation. These results indicated that Rep, TrAP and βC1 proteins of CLCuKoV-CLCuMuB can re-activate the expression of a transcriptionally silenced GFP transgene in N. benthamiana. Although Rep, TrAP, or βC1 proteins have, for other begomoviruses or begomoviruses-betasatellites, been previously shown to have TGS suppressor activity, this is the first report demonstrating that a single begomovirus-betasatellite complex encodes three suppressors of TGS.

  19. Library siRNA-generating RNA nanosponges for gene silencing by complementary rolling circle transcription.

    PubMed

    Han, Sangwoo; Kim, Hyejin; Lee, Jong Bum

    2017-08-30

    Since the discovery of RNA interference (RNAi), small interfering RNA (siRNA) has been powerful tools for gene downregulation in biomedical applications. Despite the outstanding efficacy of siRNA, the development of a therapeutic delivery system remains a challenge owing to the instability of RNA. In this study, we describe a new method for the design of siRNA-generating nanosponges by using complementary rolling circle transcription (cRCT), a technique that requires two complementary circular DNA. The sequences of one of the circular DNA are designed to have complete complementarity to the target mRNA resulting in double stranded RNA (dsRNA) that can be digested to siRNA by cellular Dicer activity. This siRNA design, called 'library siRNA', could be universally applied to fabricate RNA nanosponges targeting any known mRNA sequence.

  20. Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency.

    PubMed

    Lee, Song Hee; Caviness, Katie; Albright, Emily R; Lee, Jeong-Hee; Gelbmann, Christopher B; Rak, Mike; Goodrum, Felicia; Kalejta, Robert F

    2016-10-15

    The UL133-138 locus present in clinical strains of human cytomegalovirus (HCMV) encodes proteins required for latency and reactivation in CD34(+) hematopoietic progenitor cells and virion maturation in endothelial cells. The encoded proteins form multiple homo- and hetero-interactions and localize within secretory membranes. One of these genes, UL136 gene, is expressed as at least five different protein isoforms with overlapping and unique functions. Here we show that another gene from this locus, the UL138 gene, also generates more than one protein isoform. A long form of UL138 (pUL138-L) initiates translation from codon 1, possesses an amino-terminal signal sequence, and is a type one integral membrane protein. Here we identify a short protein isoform (pUL138-S) initiating from codon 16 that displays a subcellular localization similar to that of pUL138-L. Reporter, short-term transcription, and long-term virus production assays revealed that both pUL138-L and pUL138-S are able to suppress major immediate early (IE) gene transcription and the generation of infectious virions in cells in which HCMV latency is studied. The long form appears to be more potent at silencing IE transcription shortly after infection, while the short form seems more potent at restricting progeny virion production at later times, indicating that both isoforms of UL138 likely cooperate to promote HCMV latency. Latency allows herpesviruses to persist for the lives of their hosts in the face of effective immune control measures for productively infected cells. Controlling latent reservoirs is an attractive antiviral approach complicated by knowledge deficits for how latently infected cells are established, maintained, and reactivated. This is especially true for betaherpesviruses. The functional consequences of HCMV UL138 protein expression during latency include repression of viral IE1 transcription and suppression of virus replication. Here we show that short and long isoforms of UL138

  1. Use of the tetracycline-controlled transcriptional silencer (tTS) to eliminate transgene leak in inducible overexpression transgenic mice.

    PubMed

    Zhu, Z; Ma, B; Homer, R J; Zheng, T; Elias, J A

    2001-07-06

    The doxycycline-inducible reverse tetracycline transactivator (rtTA) is frequently used to overexpress transgenes in a temporally regulated fashion in vivo. These systems are, however, often limited by the levels of transgene expression in the absence of dox administration. The tetracycline-controlled transcriptional silencer (tTS), a fusion protein containing the tet repressor and the KRAB-AB domain of the kid-1 transcriptional repressor, is inhibited by doxycycline. We hypothesized that tTS would tighten control of transgene expression in rtTA-based systems. To test this hypothesis we generated mice in which the CC10 promoter targeted tTS to the lung, bred these mice with CC10-rtTA-interleukin 13 (IL-13) mice in which IL-13 was overexpressed in an inducible lung-specific fashion, and compared the IL-13 production and phenotypes of parental mice and the triple transgenic CC10-rtTA/tTS-IL-13 progeny of these crosses. In the CC10-rtTA-IL-13 mice, IL-13, mucus metaplasia, inflammation, alveolar enlargement, and enhanced lung volumes were noted at base line and increased greatly after doxycycline administration. In the triple transgenic tTS animals, IL-13 and the IL-13-induced phenotype could not be appreciated without doxycycline. In contrast, tTS did not alter the induction of IL-13 or the generation of the IL-13 phenotype by doxycycline. Thus, tTS effectively eliminated the baseline leak without altering the inducibility of rtTA-regulated transgenes in vivo. Optimal "off/on" regulation of transgene expression can be accomplished with the combined use of tTS and rtTA.

  2. ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo

    PubMed Central

    De La Fuente, Rabindranath; Baumann, Claudia; Viveiros, Maria M.

    2015-01-01

    A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twofold increase in transcripts for aurora kinase B, the centromeric cohesin ESCO2, DNMT1, the ubiquitin-ligase (DZIP3) and the histone methyl transferase (EHMT1). Thus, loss of ATRX activates a pathway that integrates epigenetic modifications and DNA repair in response to chromosome breaks. These results reveal the cellular response of the cleavage-stage embryo to CIN and uncover a mechanism by which centromeric fission induces the formation of large-scale chromosomal rearrangements. Our results have important implications to determine the epigenetic origins of CIN that lead to congenital birth defects and early pregnancy loss, as well as the mechanisms involved in the oocyte to embryo transition. PMID:25926359

  3. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death.

    PubMed

    Guida, Natascia; Laudati, Giusy; Anzilotti, Serenella; Secondo, Agnese; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

    2015-11-01

    Resveratrol (3,5,4'-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression.

    PubMed

    Zebec, Ziga; Zink, Isabelle Anna; Kerou, Melina; Schleper, Christa

    2016-10-13

    CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR) locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs) leading to ∼ 70-80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms.

  5. Small Ubiquitin-like Modifier (SUMO) Conjugation Impedes Transcriptional Silencing by the Polycomb Group Repressor Sex Comb on Midleg*

    PubMed Central

    Smith, Matthew; Mallin, Daniel R.; Simon, Jeffrey A.; Courey, Albert J.

    2011-01-01

    The Drosophila protein Sex Comb on Midleg (Scm) is a member of the Polycomb group (PcG), a set of transcriptional repressors that maintain silencing of homeotic genes during development. Recent findings have identified PcG proteins both as targets for modification by the small ubiquitin-like modifier (SUMO) protein and as catalytic components of the SUMO conjugation pathway. We have found that the SUMO-conjugating enzyme Ubc9 binds to Scm and that this interaction, which requires the Scm C-terminal sterile α motif (SAM) domain, is crucial for the efficient sumoylation of Scm. Scm is associated with the major Polycomb response element (PRE) of the homeotic gene Ultrabithorax (Ubx), and efficient PRE recruitment requires an intact Scm SAM domain. Global reduction of sumoylation augments binding of Scm to the PRE. This is likely to be a direct effect of Scm sumoylation because mutations in the SUMO acceptor sites in Scm enhance its recruitment to the PRE, whereas translational fusion of SUMO to the Scm N terminus interferes with this recruitment. In the metathorax, Ubx expression promotes haltere formation and suppresses wing development. When SUMO levels are reduced, we observe decreased expression of Ubx and partial haltere-to-wing transformation phenotypes. These observations suggest that SUMO negatively regulates Scm function by impeding its recruitment to the Ubx major PRE. PMID:21278366

  6. Silencing of Gonad-Inhibiting Hormone Transcripts in Litopenaeus vannamei Females by use of the RNA Interference Technology.

    PubMed

    Feijó, Rubens G; Braga, André L; Lanes, Carlos F C; Figueiredo, Márcio A; Romano, Luis A; Klosterhoff, Marta C; Nery, Luis E M; Maggioni, Rodrigo; Wasielesky, Wilson; Marins, Luis F

    2016-02-01

    The method usually employed to stimulate gonadal maturation and spawning of captive shrimp involves unilateral eyestalk ablation, which results in the removal of the endocrine complex responsible for gonad-inhibiting hormone (GIH) synthesis and release. In the present study, RNAi technology was used to inhibit transcripts of GIH in Litopenaeus vannamei females. The effect of gene silencing on gonad development was assessed by analyzing the expression of GIH and vitellogenin, respectively, in the eyestalk and ovaries of L. vannamei females, following ablation or injection with dsRNA-GIH, dsRNA-IGSF4D (non-related dsRNA), or saline solution. Histological analyses were performed to determine the stage of gonadal development and to assess the diameter of oocytes throughout the experimental procedure. Only oocytes at pre-vitellogenesis and primary vitellogenesis stages were identified in females injected with dsRNA-GIH, dsRNA-IGSF4D, or saline solution. Oocytes at all developmental stages were observed in eyestalk-ablated females, with predominance of later stages, such as secondary vitellogenesis and mature oocytes. Despite achieving 64, 73, and 71% knockdown of eyestalk GIH mRNA levels by 15, 30, and 37 days post-injection (dpi), respectively, in dsRNA-GIH-injected females, the expected increase in ovary vitellogenin mRNA expression was only observed on the 37th dpi. This is the first report of the use of RNAi technology to develop an alternative method to eyestalk ablation in captive L. vannamei shrimps.

  7. Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

    PubMed Central

    Zebec, Ziga; Zink, Isabelle Anna; Kerou, Melina; Schleper, Christa

    2016-01-01

    CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR) locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs) leading to ∼ 70–80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms. PMID:27507792

  8. Transient Reversal of Episome Silencing Precedes VP16-Dependent Transcription during Reactivation of Latent HSV-1 in Neurons

    PubMed Central

    Kim, Ju Youn; Mandarino, Angelo; Chao, Moses V.; Mohr, Ian; Wilson, Angus C.

    2012-01-01

    Herpes simplex virus type-1 (HSV-1) establishes latency in peripheral neurons, creating a permanent source of recurrent infections. The latent genome is assembled into chromatin and lytic cycle genes are silenced. Processes that orchestrate reentry into productive replication (reactivation) remain poorly understood. We have used latently infected cultures of primary superior cervical ganglion (SCG) sympathetic neurons to profile viral gene expression following a defined reactivation stimulus. Lytic genes are transcribed in two distinct phases, differing in their reliance on protein synthesis, viral DNA replication and the essential initiator protein VP16. The first phase does not require viral proteins and has the appearance of a transient, widespread de-repression of the previously silent lytic genes. This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron. During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters. The transactivation function supplied by VP16 promotes increased viral lytic gene transcription leading to the onset of genome amplification and the production of infectious viral particles. Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons. PMID:22383875

  9. Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena

    PubMed Central

    Schoeberl, Ursula E.; Kurth, Henriette M.; Noto, Tomoko; Mochizuki, Kazufumi

    2012-01-01

    The ciliated protozoan Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. DNA elimination is epigenetically controlled by DNA sequences of the parental macronuclear genome, and this epigenetic regulation is mediated by small RNAs (scan RNAs [scnRNAs]) of ∼28–30 nucleotides that are produced and function by an RNAi-related mechanism. Here, we examine scnRNA production and turnover by deep sequencing. scnRNAs are produced exclusively from the micronucleus and nonhomogeneously from a variety of chromosomal locations. scnRNAs are preferentially derived from the eliminated sequences, and this preference is mainly determined at the level of transcription. Despite this bias, a significant fraction of scnRNAs is also derived from the macronuclear-destined sequences, and these scnRNAs are degraded during the course of sexual reproduction. These results indicate that the pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and the selective degradation of scnRNAs in the parental macronucleus. PMID:22855833

  10. The analysis of novel distal Cebpa enhancers and silencers using a transcriptional model reveals the complex regulatory logic of hematopoietic lineage specification.

    PubMed

    Bertolino, Eric; Reinitz, John; Manu

    2016-05-01

    C/EBPα plays an instructive role in the macrophage-neutrophil cell-fate decision and its expression is necessary for neutrophil development. How Cebpa itself is regulated in the myeloid lineage is not known. We decoded the cis-regulatory logic of Cebpa, and two other myeloid transcription factors, Egr1 and Egr2, using a combined experimental-computational approach. With a reporter design capable of detecting both distal enhancers and silencers, we analyzed 46 putative cis-regulatory modules (CRMs) in cells representing myeloid progenitors, and derived early macrophages or neutrophils. In addition to novel enhancers, this analysis revealed a surprisingly large number of silencers. We determined the regulatory roles of 15 potential transcriptional regulators by testing 32,768 alternative sequence-based transcriptional models against CRM activity data. This comprehensive analysis allowed us to infer the cis-regulatory logic for most of the CRMs. Silencer-mediated repression of Cebpa was found to be effected mainly by TFs expressed in non-myeloid lineages, highlighting a previously unappreciated contribution of long-distance silencing to hematopoietic lineage resolution. The repression of Cebpa by multiple factors expressed in alternative lineages suggests that hematopoietic genes are organized into densely interconnected repressive networks instead of hierarchies of mutually repressive pairs of pivotal TFs. More generally, our results demonstrate that de novo cis-regulatory dissection is feasible on a large scale with the aid of transcriptional modeling. Current address: Department of Biology, University of North Dakota, 10 Cornell Street, Stop 9019, Grand Forks, ND 58202-9019, USA.

  11. Mixture models reveal multiple positional bias types in RNA-Seq data and lead to accurate transcript concentration estimates.

    PubMed

    Tuerk, Andreas; Wiktorin, Gregor; Güler, Serhat

    2017-05-01

    Accuracy of transcript quantification with RNA-Seq is negatively affected by positional fragment bias. This article introduces Mix2 (rd. "mixquare"), a transcript quantification method which uses a mixture of probability distributions to model and thereby neutralize the effects of positional fragment bias. The parameters of Mix2 are trained by Expectation Maximization resulting in simultaneous transcript abundance and bias estimates. We compare Mix2 to Cufflinks, RSEM, eXpress and PennSeq; state-of-the-art quantification methods implementing some form of bias correction. On four synthetic biases we show that the accuracy of Mix2 overall exceeds the accuracy of the other methods and that its bias estimates converge to the correct solution. We further evaluate Mix2 on real RNA-Seq data from the Microarray and Sequencing Quality Control (MAQC, SEQC) Consortia. On MAQC data, Mix2 achieves improved correlation to qPCR measurements with a relative increase in R2 between 4% and 50%. Mix2 also yields repeatable concentration estimates across technical replicates with a relative increase in R2 between 8% and 47% and reduced standard deviation across the full concentration range. We further observe more accurate detection of differential expression with a relative increase in true positives between 74% and 378% for 5% false positives. In addition, Mix2 reveals 5 dominant biases in MAQC data deviating from the common assumption of a uniform fragment distribution. On SEQC data, Mix2 yields higher consistency between measured and predicted concentration ratios. A relative error of 20% or less is obtained for 51% of transcripts by Mix2, 40% of transcripts by Cufflinks and RSEM and 30% by eXpress. Titration order consistency is correct for 47% of transcripts for Mix2, 41% for Cufflinks and RSEM and 34% for eXpress. We, further, observe improved repeatability across laboratory sites with a relative increase in R2 between 8% and 44% and reduced standard deviation.

  12. Overcoming H-NS-mediated Transcriptional Silencing of Horizontally Acquired Genes by the PhoP and SlyA Proteins in Salmonella enterica*S⃞

    PubMed Central

    Perez, J. Christian; Latifi, Tammy; Groisman, Eduardo A.

    2008-01-01

    The acquisition of new traits through horizontal gene transfer depends on the ability of the recipient organism to express the incorporated genes. However, foreign DNA appears to be silenced by the histone-like nucleoid-structuring protein (H-NS) in several enteric pathogens, raising the question of how this silencing is overcome and the acquired genes are expressed at the right time and place. To address this question, we investigated transcription of the horizontally acquired ugtL and pagC genes from Salmonella enterica, which is dependent on the regulatory DNA-binding proteins PhoP and SlyA. We reconstituted transcription of the ugtL and pagC genes in vitro and determined occupancy of their respective promoters by PhoP, H-NS, and RNA polymerase in vivo. The SlyA protein counteracted H-NS-promoted repression in vitro but could not promote gene transcription by itself. PhoP-promoted transcription required SlyA when H-NS was present but not in its absence. In vivo, H-NS remained bound to the ugtL and pagC promoters under inducing conditions that promoted RNA polymerase recruitment and transcription of the ugtL and pagC genes. Our results indicate that relief of H-NS repression and recruitment of RNA polymerase are controlled by different regulatory proteins that act in concert to express horizontally acquired genes. PMID:18270203

  13. Expression Profiles and RNAi Silencing of Inhibitor of Apoptosis Transcripts in Aedes, Anopheles, and Culex Mosquitoes (Diptera: Culicidae).

    PubMed

    Puglise, Jason M; Estep, Alden S; Becnel, James J

    2016-03-01

    Effective mosquito control is vital to curtail the devastating health effects of many vectored diseases. RNA interference (RNAi)-mediated control of mosquitoes is an attractive alternative to conventional chemical pesticides. Previous studies have suggested that transcripts for inhibitors of apoptosis (IAPs) may be good RNAi targets. To revisit and extend previous reports, we examined the expression of Aedes aegypti (L.) IAPs (AaeIAPs) 1, 2, 5, 6, 9, and a viral IAP-associated factor (vIAF) as well as Anopheles quadrimaculatus Say and Culex quinquefasciatus Say IAP1 homologs (AquIAP1 and CquIAP1) in adult females. Expression profiles of IAPs suggested that some older female mosquitoes had significantly higher IAP mRNA levels when compared to the youngest ones. Minor differences in expression of AaeIAPs were observed in mosquitoes that imbibed a bloodmeal, but the majority of the time points (up to 48 h) were not significantly different. Although in vitro experiments with the Ae. aegypti Aag-2 cell line demonstrated that the various AaeIAPs could be effectively knocked down within one day after dsRNA treatment, only Aag-2 cells treated with dsIAP1 displayed apoptotic morphology. Gene silencing and mortality were also evaluated after topical application and microinjection of the same dsRNAs into female Ae. aegypti. In contrast to previous reports, topical administration of dsRNA against AaeIAP1 did not yield a significant reduction in gene expression or increased mortality. Knockdown of IAP1 and other IAPs by microinjection did not result in significant mortality. In toto, our findings suggest that IAPs may not be suitable RNAi targets for controlling adult mosquito populations.

  14. GC-compositional strand bias around transcription start sites in plants and fungi

    PubMed Central

    Fujimori, Shigeo; Washio, Takanori; Tomita, Masaru

    2005-01-01

    Background A GC-compositional strand bias or GC-skew (=(C-G)/(C+G)), where C and G denote the numbers of cytosine and guanine residues, was recently reported near the transcription start sites (TSS) of Arabidopsis genes. However, it is unclear whether other eukaryotic species have equally prominent GC-skews, and the biological meaning of this trait remains unknown. Results Our study confirmed a significant GC-skew (C > G) in the TSS of Oryza sativa (rice) genes. The full-length cDNAs and genomic sequences from Arabidopsis and rice were compared using statistical analyses. Despite marked differences in the G+C content around the TSS in the two plants, the degrees of bias were almost identical. Although slight GC-skew peaks, including opposite skews (C < G), were detected around the TSS of genes in human and Drosophila, they were qualitatively and quantitatively different from those identified in plants. However, plant-like GC-skew in regions upstream of the translation initiation sites (TIS) in some fungi was identified following analyses of the expressed sequence tags and/or genomic sequences from other species. On the basis of our dataset, we estimated that >70 and 68% of Arabidopsis and rice genes, respectively, had a strong GC-skew (>0.33) in a 100-bp window (that is, the number of C residues was more than double the number of G residues in a +/-100-bp window around the TSS). The mean GC-skew value in the TSS of highly-expressed genes in Arabidopsis was significantly greater than that of genes with low expression levels. Many of the GC-skew peaks were preferentially located near the TSS, so we examined the potential value of GC-skew as an index for TSS identification. Our results confirm that the GC-skew can be used to assist the TSS prediction in plant genomes. Conclusion The GC-skew (C > G) around the TSS is strictly conserved between monocot and eudicot plants (ie. angiosperms in general), and a similar skew has been observed in some fungi. Highly

  15. Biallelic insertion of a transcriptional terminator via the CRISPR/Cas9 system efficiently silences expression of protein-coding and non-coding RNA genes.

    PubMed

    Liu, Yangyang; Han, Xiao; Yuan, Junting; Geng, Tuoyu; Chen, Shihao; Hu, Xuming; Cui, Isabelle H; Cui, Hengmi

    2017-04-07

    The type II bacterial CRISPR/Cas9 system is a simple, convenient, and powerful tool for targeted gene editing. Here, we describe a CRISPR/Cas9-based approach for inserting a poly(A) transcriptional terminator into both alleles of a targeted gene to silence protein-coding and non-protein-coding genes, which often play key roles in gene regulation but are difficult to silence via insertion or deletion of short DNA fragments. The integration of 225 bp of bovine growth hormone poly(A) signals into either the first intron or the first exon or behind the promoter of target genes caused efficient termination of expression of PPP1R12C, NSUN2 (protein-coding genes), and MALAT1 (non-protein-coding gene). Both NeoR and PuroR were used as markers in the selection of clonal cell lines with biallelic integration of a poly(A) signal. Genotyping analysis indicated that the cell lines displayed the desired biallelic silencing after a brief selection period. These combined results indicate that this CRISPR/Cas9-based approach offers an easy, convenient, and efficient novel technique for gene silencing in cell lines, especially for those in which gene integration is difficult because of a low efficiency of homology-directed repair.

  16. Identification of a class of human cancer germline genes with transcriptional silencing refractory to the hypomethylating drug 5-aza-2′-deoxycytidine.

    PubMed Central

    Almatrafi, Ahmed; Feichtinger, Julia; Vernon, Ellen G.; Escobar, Natalia Gomez; Wakeman, Jane A.; Larcombe, Lee D.; McFarlane, Ramsay J.

    2014-01-01

    Bona fide germline genes have expression restricted to the germ cells of the gonads. Testis-specific germline development-associated genes can become activated in cancer cells and can potentially drive the oncogenic process and serve as therapeutic/biomarker targets; such germline genes are referred to as cancer/testis genes. Many cancer/testis genes are silenced via hypermethylation of CpG islands in their associated transcriptional control regions and become activated upon treatment with DNA hypomethylating agents; such hypomethylation-induced activation of cancer/testis genes provides a potential combination approach to augment immunotherapeutics. Thus, understanding cancer/testis gene regulation is of increasing clinical importance. Previously studied cancer/testis gene activation has focused on X chromosome encoded cancer/testis genes. Here we find that a sub-set of non-X encoded cancer/testis genes are silenced in non-germline cells via a mechanism that is refractory to epigenetic dysregulation, including treatment with the hypomethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor tricostatin A. These findings formally indicate that there is a sub-group of the clinically important cancer/testis genes that are unlikely to be activated in clinical therapeutic approaches using hypomethylating agents and it indicates a unique transcriptional silencing mechanism for germline genes in non-germline cells that might provide a target mechanism for new clinical therapies. PMID:25594001

  17. A Novel Repeat-Associated Small Interfering RNA-Mediated Silencing Pathway Downregulates Complementary Sense gypsy Transcripts in Somatic Cells of the Drosophila Ovary▿

    PubMed Central

    Pélisson, Alain; Sarot, Emeline; Payen-Groschêne, Geneviève; Bucheton, Alain

    2007-01-01

    Replication of the gypsy endogenous retrovirus involves contamination of the female germ line by adjacent somatic tissues. This is prevented by flam, an as-yet-uncloned heterochromatic pericentromeric locus, at the level of transcript accumulation in these somatic ovarian tissues. We tested the effect of a presumptive RNA silencing mechanism on the accumulation of RNAs produced by constructs containing various gypsy sequences and report that the efficiency of silencing is indeed correlated with the amount of complementary RNAs, 25 to 30 nucleotides in length, in the ovary. For instance, while these RNAs were found to display a three- to fivefold excess of the antisense strands, only the transcripts that contain the complementary sense gypsy sequences could be repressed, indicating that they are targeted at the RNA, not DNA, level. Their size and asymmetry in strand polarity are typical of the novel repeat-associated small interfering RNA (rasiRNA)-mediated pathway, recently suspected to prevent the deleterious expression of selfish DNA specifically in the germ line. Unlike microRNAs (but like rasiRNAs and, surprisingly, siRNAs as well), gypsy rasiRNAs are modified at the 3′ end. The rasiRNA-associated protein Piwi (but not Aub) is required for gypsy silencing, whereas Dicer-2 (which makes siRNAs) is not. In contrast, piwi, aub, and flam do not appear to affect somatic siRNA-mediated silencing. The amount of gypsy rasiRNAs is genetically determined by the flam locus in a provirus copy number-independent manner and is triggered in the somatic tissues by some pericentromeric provirus(es), which are thereby able to protect the germ line from retroviral invasion. PMID:17135323

  18. Signal Transducer and Activator of Transcription 1 (STAT1) is Essential for Chromium Silencing of Gene Induction in Human Airway Epithelial Cells

    PubMed Central

    Nemec, Antonia A.; Barchowsky, Aaron

    2009-01-01

    Hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms that may involve the silencing of inducible protective genes. The current study investigated the hypothesis that Cr(VI) actively signals through a signal transducer and activator of transcription 1 (STAT1)–dependent pathway to silence nickel (Ni)–induced expression of vascular endothelial cell growth factor A (VEGFA), an important mediator of lung injury and repair. In human bronchial airway epithelial (BEAS-2B) cells, Ni-induced VEGFA transcription by stimulating an extracellular regulated kinase (ERK) signaling cascade that involved Src kinase–activated Sp1 transactivation, as well as increased hypoxia-inducible factor-1α (HIF-1α) stabilization and DNA binding. Ni-stimulated ERK, Src, and HIF-1α activities, as well as Ni-induced VEGFA transcript levels were inhibited in Cr(VI)-exposed cells. We previously demonstrated that Cr(VI) stimulates STAT1 to suppress VEGFA expression. In BEAS-2B cells stably expressing STAT1 short hairpin RNA, Cr(VI) increased VEGFA transcript levels and Sp1 transactivation. Moreover, in the absence of STAT1, Cr(VI), and Ni coexposures positively interacted to further increase VEGFA transcripts. This study demonstrates that metal-stimulated signaling cascades interact to regulate transcription and induction of adaptive or repair responses in airway cells. In addition, the data implicate STAT1 as a rate limiting mediator of Cr(VI)-stimulated gene regulation and suggest that cells lacking STAT1, such as many tumor cell lines, have opposite responses to Cr(VI) relative to normal cells. PMID:19403854

  19. Transcriptional Repression and RNA Silencing Act Synergistically To Demonstrate the Function of the Eleventh Component of the Vaccinia Virus Entry-Fusion Complex

    PubMed Central

    Wolfe, Cindy L.; Ojeda, Suany

    2012-01-01

    Poxviruses have an elaborate system for infecting cells comprising several proteins for attachment and a larger number dedicated to membrane fusion and entry. Thus far, 11 proteins have been identified as components of the vaccinia virus (VACV) entry-fusion complex (EFC), and 10 of these proteins have been shown to be required for entry. J5, the remaining functionally uncharacterized component of the complex, is conserved in all poxviruses, has a predicted C-terminal transmembrane domain, and is an N-terminally truncated paralog of two other EFC proteins. To determine the role of J5, we constructed a mutant that inducibly regulates J5 transcription. Although the virus yield was reduced only about 80% without inducer, the inability to isolate a J5 deletion mutant suggested an essential function. To enhance stringency, we employed RNA silencing alone and together with transcriptional repression of the inducible mutant. The yield of infectious virus was reduced 4- to 5-fold by repression, 2-fold by silencing, and 60-fold by the combination of the two. Virus particles made under the latter conditions appeared to contain a full complement of proteins excluding J5 but had very low infectivity. Further studies indicated that after binding to cells, J5-deficient virions had a defect in core entry and an inability to induce syncytium formation. In addition, we confirmed that J5 is associated with the EFC by affinity purification. These data indicate that J5 is a functional component of the EFC and highlights the advantage of combining transcriptional repression and RNA silencing for stringent reduction of gene expression. PMID:22013036

  20. Virus-Induced Gene Silencing Identifies an Important Role of the TaRSR1 Transcription Factor in Starch Synthesis in Bread Wheat.

    PubMed

    Liu, Guoyu; Wu, Yufang; Xu, Mengjun; Gao, Tian; Wang, Pengfei; Wang, Lina; Guo, Tiancai; Kang, Guozhang

    2016-09-23

    The function of a wheat starch regulator 1 (TaRSR1) in regulating the synthesis of grain storage starch was determined using the barley stripe mosaic virus-virus induced gene-silencing (BSMV-VIGS) method in field experiments. Chlorotic stripes appeared on the wheat spikes infected with barley stripe mosaic virus-virus induced gene-silencing- wheat starch regulator 1 (BSMV-VIGS-TaRSR1) at 15 days after anthesis, at which time the transcription levels of the TaRSR1 gene significantly decreased. Quantitative real-time PCR was also used to measure the transcription levels of 26 starch synthesis-related enzyme genes in the grains of BSMV-VIGS-TaRSR1-silenced wheat plants at 20, 27, and 31 days after anthesis. The results showed that the transcription levels of some starch synthesis-related enzyme genes were markedly induced at different sampling time points: TaSSI, TaSSIV, TaBEIII, TaISA1, TaISA3, TaPHOL, and TaDPE1 genes were induced at each of the three sampling time points and TaAGPS1-b, TaAGPL1, TaAGPL2, TaSSIIb, TaSSIIc, TaSSIIIb, TaBEI, TaBEIIa, TaBEIIb, TaISA2, TaPHOH, and TaDPE2 genes were induced at one sampling time point. Moreover, both the grain starch contents, one thousand kernel weights, grain length and width of BSMV-VIGS-TaRSR1-infected wheat plants significantly increased. These results suggest that TaRSR1 acts as a negative regulator and plays an important role in starch synthesis in wheat grains by temporally regulating the expression of specific starch synthesis-related enzyme genes.

  1. A high-throughput virus-induced gene-silencing vector for screening transcription factors in virus-induced plant defense response in orchid.

    PubMed

    Lu, Hsiang-Chia; Hsieh, Ming-Hsien; Chen, Cheng-En; Chen, Hong-Hwa; Wang, Hsiang-Iu; Yeh, Hsin-Hung

    2012-06-01

    The large number of species and worldwide spread of species of Orchidaceae indicates their successful adaptation to environmental stresses. Thus, orchids provide rich resources to study how plants have evolved to cope with stresses. This report describes our improvement of our previously reported orchid virus-induced gene silencing vector, pCymMV-pro60, with a modified Gateway cloning system which requires only one recombination and can be inoculated by agroinfiltration. We cloned 1,700 DNA fragments, including 187 predicted transcription factors derived from an established expression sequence tag library of orchid, into pCymMV-Gateway. Phalaenopsis aphrodite was inoculated with these vectors that contained DNA fragments of the 187 predicted transcription factors. The viral vector initially triggered the expression of the salicylic acid (SA)-related plant defense responses and later induced silencing of the endogenous target transcription factor genes. By monitoring the expression of the SA-related plant defense marker PhaPR1 (homolog of PR1), we identified a gene, PhaTF15, involved in the expression of PhaPR1. Knockdown of PhaTF15 by virus-induced gene silencing and by transient delivery of double-stranded RNA (dsRNA) reduced expression of the orchid homolog of the conserved positive defense regulator NPR1, PhaNPR1. Cymbidium mosaic virus also accumulated to high levels with knockdown of PhaTF15 by transient delivery of dsRNA. We demonstrated efficient cloning and screening strategies for high-throughput analysis of orchid and identify a gene, PhaTF15, involved in regulation of SA-related plant defense.

  2. Virus-Induced Gene Silencing Identifies an Important Role of the TaRSR1 Transcription Factor in Starch Synthesis in Bread Wheat

    PubMed Central

    Liu, Guoyu; Wu, Yufang; Xu, Mengjun; Gao, Tian; Wang, Pengfei; Wang, Lina; Guo, Tiancai; Kang, Guozhang

    2016-01-01

    The function of a wheat starch regulator 1 (TaRSR1) in regulating the synthesis of grain storage starch was determined using the barley stripe mosaic virus—virus induced gene-silencing (BSMV-VIGS) method in field experiments. Chlorotic stripes appeared on the wheat spikes infected with barley stripe mosaic virus-virus induced gene-silencing- wheat starch regulator 1 (BSMV-VIGS-TaRSR1) at 15 days after anthesis, at which time the transcription levels of the TaRSR1 gene significantly decreased. Quantitative real-time PCR was also used to measure the transcription levels of 26 starch synthesis-related enzyme genes in the grains of BSMV-VIGS-TaRSR1-silenced wheat plants at 20, 27, and 31 days after anthesis. The results showed that the transcription levels of some starch synthesis-related enzyme genes were markedly induced at different sampling time points: TaSSI, TaSSIV, TaBEIII, TaISA1, TaISA3, TaPHOL, and TaDPE1 genes were induced at each of the three sampling time points and TaAGPS1-b, TaAGPL1, TaAGPL2, TaSSIIb, TaSSIIc, TaSSIIIb, TaBEI, TaBEIIa, TaBEIIb, TaISA2, TaPHOH, and TaDPE2 genes were induced at one sampling time point. Moreover, both the grain starch contents, one thousand kernel weights, grain length and width of BSMV-VIGS-TaRSR1-infected wheat plants significantly increased. These results suggest that TaRSR1 acts as a negative regulator and plays an important role in starch synthesis in wheat grains by temporally regulating the expression of specific starch synthesis-related enzyme genes. PMID:27669224

  3. Protective effect of caffeine against high sugar-induced transcription of microRNAs and consequent gene silencing: a study using lenses of galactosemic mice.

    PubMed

    Varma, Shambhu D; Kovtun, Svitlana

    2013-01-01

    Previous studies have shown that caffeine prevents the formation of cataracts induced by a high-galactose diet and consequent oxidative stress. The objective of this study was to investigate if this protective effect is reflected in the attenuation of the transcription of microRNAs (miRNAs) known to induce apoptosis and cell death by gene silencing. Young CD-1 mice were fed either a normal laboratory diet or a diet containing 25% galactose with or without 1% caffeine. One week later, the animals were euthanized, and the lenses isolated and promptly processed for RNA isolation and subsequent preparation of cDNAs by reverse transcriptase reaction. Mature miRNA (miR)-specific cDNAs were then quantified with PCR in a 96-well microRNA-specific cassette using an ABI7900HT PCR machine. As expected from previous studies, the lenses were positive for all 84 miRs corresponding to the miRNA probes present in the cassette wells. However, the levels of at least 19 miRs were significantly elevated in galactosemic lenses compared to those in the normal lenses. The majority are proapoptotic. Such elevation was inhibited by caffeine. This has been demonstrated for the first time. Since aberrant elevation of miRNAs silences various genes and consequently deactivates protein translation, and since caffeine downregulates such aberration, the beneficial effect of caffeine could be attributed to its ability to suppress elevation of toxic miRs and consequent gene silencing.

  4. Transcriptional silencing of N-Myc downstream-regulated gene 1 (NDRG1) in metastatic colon cancer cell line SW620.

    PubMed

    Li, Qian; Chen, Hong

    2011-02-01

    N-Myc downstream-regulated gene 1 (NDRG1) plays vital roles in tumor metastasis suppression and is frequently silenced in metastatic colon cancers. NDRG1 is silenced in a highly metastatic colon cancer cell line SW620. The objective of this study was to investigate the potential mechanisms involved in silencing of the NDRG1 gene. SW480 and SW620 are two colon cancer cell lines established from the same patient with different metastatic potentials, making them an ideal model for investigation of metastatic mechanisms. Knockdown of NDRG1 in SW480 to a level that is similar to that in SW620 also modulated cell cycle and proliferation in SW480 towards the status of the highly metastatic SW620. Epigenetic mechanisms of the transcriptional control of NDRG1 were investigated. The silencing of NDRG1 in SW620 was not due to promoter hyper-methylation as bisulfite sequencing of the NDRG1 promoter showed minimal DNA methylation in both cell lines. On the other hand, chromatin immunoprecipitation showed a significantly higher level of RNA polymerase II (Pol II) association with the NDRG1 promoter in SW480 compared to SW620, in agreement with its gene expression level. The low Pol II binding at the NDRG1 promoter in SW620 was associated with gene-wide decrease in histone H4 acetylation and increase in histone H3 serine 10 phosphorylation. Meanwhile, the NDRG1 coding region showed much higher histone H3 lysine 4 methylation in SW480. In conclusion we observed unique histone modifications in two colon cancer cell lines with different metastatic potentials, indicating possible mechanisms for the down-regulation of NDRG1 in metastatic SW620.

  5. Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

    SciTech Connect

    Lv, Peng; Xue, Peng; Dong, Jian; Peng, Hui; Clewell, Rebecca; Wang, Aiping; Wang, Yue; Peng, Shuangqing; Qu, Weidong; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

    2013-11-01

    Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation.

  6. Silencing of the transcription factor STAT3 sensitizes lung cancer cells to DNA damaging drugs, but not to TNFα- and NK cytotoxicity

    SciTech Connect

    Kulesza, Dorota W.; Carré, Thibault; Chouaib, Salem; Kaminska, Bozena

    2013-02-15

    Transcription factor STAT3 (Signal Transducers and Activators of Transcription 3) is persistently active in human tumors and may contribute to tumor progression. Inhibition of STAT3 expression/activity could be a good strategy to modulate tumor cell survival and responses to cancer chemotherapeutics or immune cytotoxicity. We silenced STAT3 expression in human A549 lung cancer cells to elucidate its role in cell survival and resistance to chemotherapeutics, TNFα and natural killer (NK)-mediated cytotoxicity. We demonstrate that STAT3 is not essential for basal survival and proliferation of A549 cancer cells. Stable silencing of STAT3 expression sensitized A549 cells to DNA damaging chemotherapeutics doxorubicin and cisplatin in a p53-independent manner. Sensitization to DNA damage-inducing chemotherapeutics could be due to down-regulation of the Bcl-xL expression in STAT3 depleted cells. In contrast, knockdown of STAT3 in cancer cells did not modulate responses to TNFα and NK-mediated cytotoxicity. We found that STAT3 depletion increased the NFκB activity likely providing the compensatory, pro-survival signal. The treatment with TNFα, but not doxorubicin, enhanced this effect. We conclude that STAT3 is not crucial for the control of basal cell proliferation and survival of lung carcinoma cells but modulates susceptibility to DNA damaging chemotherapeutics by regulation of intrinsic pro-survival pathways. - Highlights: ► STAT3 silencing is negligent for basal lung cancer cell viability and proliferation. ► STAT3 depletion sensitizes lung cancer cells to DNA damaging chemotherapeutics. ► STAT3 depletion has no effect on susceptibility to extrinsic apoptosis inducers. ► Increased pro-survival NFκB activity may compensate for STAT3 depletion.

  7. Tobacco streak virus (strain dahlia) suppresses post-transcriptional gene silencing of flavone synthase II in black dahlia cultivars and causes a drastic flower color change.

    PubMed

    Deguchi, Ayumi; Tatsuzawa, Fumi; Hosokawa, Munetaka; Doi, Motoaki; Ohno, Sho

    2015-09-01

    Tobacco streak virus suppressed post-transcriptional gene silencing and caused a flower color change in black dahlias, which supported the role of cyanidin-based anthocyanins for black flower appearance. Black flower color of dahlia (Dahlia variabilis) has been attributed, in part, to the high accumulation of cyanidin-based anthocyanins that occurs when flavone synthesis is reduced because of post-transcriptional gene silencing (PTGS) of flavone synthase II (DvFNS). There are also purple-flowering plants that have emerged from a black cultivar 'Kokucho'. We report that the purple color is not caused by a mutation, as previously thought, but by infection with tobacco streak virus (TSVdahlia), which suppresses the PTGS of DvFNS. When TSVdahlia was eliminated from the purple-flowering 'Kokucho' by leaf primordia-free shoot apical meristem culture, the resulting flowers were black. TSVdahlia-infected purple flowers had lower numbers of siRNAs to DvFNS than black flowers, suggesting that TSVdahlia has a silencing suppressor. The graft inoculation of other black cultivars with TSVdahlia altered their flower color drastically except for 'Fidalgo Blacky', a very deep black cultivar with the highest amount of cyanidin-based anthocyanins. The flowers of all six TSVdahlia-infected cultivars accumulated increased amounts of flavones and reduced amounts of cyanidin-based anthocyanins. 'Fidalgo Blacky' remained black despite the change in pigment accumulation, and the amounts of cyanidin-based anthocyanins in its TSVdahlia-infected plants were still higher than those of other cultivars. We propose that black flower color in dahlia is controlled by two different mechanisms that increase the amount of cyanidin-based anthocyanins: DvFNS PTGS-dependent and -independent mechanisms. If both mechanisms occur simultaneously, the flower color will be blacker than if only a single mechanism is active.

  8. The Cytosolic Iron-Sulfur Cluster Assembly Protein MMS19 Regulates Transcriptional Gene Silencing, DNA Repair, and Flowering Time in Arabidopsis

    PubMed Central

    Han, Yong-Feng; Huang, Huan-Wei; Li, Lin; Cai, Tao; Chen, She; He, Xin-Jian

    2015-01-01

    MMS19 is an essential component of the cytoplasmic iron-sulfur (Fe-S) cluster assembly complex in fungi and mammals; the mms19 null mutant alleles are lethal. Our study demonstrates that MMS19/MET18 in Arabidopsis thaliana interacts with the cytoplasmic Fe-S cluster assembly complex but is not an essential component of the complex. We find that MMS19 also interacts with the catalytic subunits of DNA polymerases, which have been demonstrated to be involved in transcriptional gene silencing (TGS), DNA repair, and flowering time regulation. Our results indicate that MMS19 has a similar biological function, suggesting a functional link between MMS19 and DNA polymerases. In the mms19 null mutant, the assembly of Fe-S clusters on the catalytic subunit of DNA polymerase α is reduced but not blocked, which is consistent with the viability of the mutant. Our study suggests that MMS19 assists the assembly of Fe-S clusters on DNA polymerases in the cytosol, thereby facilitating transcriptional gene silencing, DNA repair, and flowering time control. PMID:26053632

  9. "The Silence Itself Is Enough of a Statement": The Day of Silence and LGBTQ Awareness Raising

    ERIC Educational Resources Information Center

    Woolley, Susan W.

    2012-01-01

    This ethnographic study of a high school gay-straight alliance club examines unintended consequences of silence during the Day of Silence, a day of action aimed at addressing anti-LGBTQ bias in schools. While this strategy calls for students to engage in intentional silences to raise awareness of anti-LGBTQ bias, it does not necessarily lead…

  10. "The Silence Itself Is Enough of a Statement": The Day of Silence and LGBTQ Awareness Raising

    ERIC Educational Resources Information Center

    Woolley, Susan W.

    2012-01-01

    This ethnographic study of a high school gay-straight alliance club examines unintended consequences of silence during the Day of Silence, a day of action aimed at addressing anti-LGBTQ bias in schools. While this strategy calls for students to engage in intentional silences to raise awareness of anti-LGBTQ bias, it does not necessarily lead…

  11. GW182-Free microRNA Silencing Complex Controls Post-transcriptional Gene Expression during Caenorhabditis elegans Embryogenesis

    PubMed Central

    Jannot, Guillaume; Michaud, Pascale; Quévillon Huberdeau, Miguel; Morel-Berryman, Louis; Brackbill, James A.; McJunkin, Katherine; Nakanishi, Kotaro; Simard, Martin J.

    2016-01-01

    MicroRNAs and Argonaute form the microRNA induced silencing complex or miRISC that recruits GW182, causing mRNA degradation and/or translational repression. Despite the clear conservation and molecular significance, it is unknown if miRISC-GW182 interaction is essential for gene silencing during animal development. Using Caenorhabditis elegans to explore this question, we examined the relationship and effect on gene silencing between the GW182 orthologs, AIN-1 and AIN-2, and the microRNA-specific Argonaute, ALG-1. Homology modeling based on human Argonaute structures indicated that ALG-1 possesses conserved Tryptophan-binding Pockets required for GW182 binding. We show in vitro and in vivo that their mutations severely altered the association with AIN-1 and AIN-2. ALG-1 tryptophan-binding pockets mutant animals retained microRNA-binding and processing ability, but were deficient in reporter silencing activity. Interestingly, the ALG-1 tryptophan-binding pockets mutant phenocopied the loss of alg-1 in worms during larval stages, yet was sufficient to rescue embryonic lethality, indicating the dispensability of AINs association with the miRISC at this developmental stage. The dispensability of AINs in miRNA regulation is further demonstrated by the capacity of ALG-1 tryptophan-binding pockets mutant to regulate a target of the embryonic mir-35 microRNA family. Thus, our results demonstrate that the microRNA pathway can act independently of GW182 proteins during C. elegans embryogenesis. PMID:27935964

  12. Transcription of AAT•ATT Triplet Repeats in Escherichia coli Is Silenced by H-NS and IS1E Transposition

    PubMed Central

    Pan, Xuefeng; Liao, Lingni; Yang, Li; Li, Hongquan

    2010-01-01

    Background The trinucleotide repeats AAT•ATT are simple DNA sequences that potentially form different types of non-B DNA secondary structures and cause genomic instabilities in vivo. Methodology and Principal Findings The molecular mechanism underlying the maintenance of a 24-triplet AAT•ATT repeat was examined in E.coli by cloning the repeats into the EcoRI site in plasmid pUC18 and into the attB site on the E.coli genome. Either the AAT or the ATT strand acted as lagging strand template in a replication fork. Propagations of the repeats in either orientation on plasmids did not affect colony morphology when triplet repeat transcription using the lacZ promoter was repressed either by supplementing LacIQ in trans or by adding glucose into the medium. In contrast, transparent colonies were formed by inducing transcription of the repeats, suggesting that transcription of AAT•ATT repeats was toxic to cell growth. Meanwhile, significant IS1E transposition events were observed both into the triplet repeats region proximal to the promoter side, the promoter region of the lacZ gene, and into the AAT•ATT region itself. Transposition reversed the transparent colony phenotype back into healthy, convex colonies. In contrast, transcription of an 8-triplet AAT•ATT repeat in either orientation on plasmids did not produce significant changes in cell morphology and did not promote IS1E transposition events. We further found that a role of IS1E transposition into plasmids was to inhibit transcription through the repeats, which was influenced by the presence of the H-NS protein, but not of its paralogue StpA. Conclusions and Significance Our findings thus suggest that the longer AAT•ATT triplet repeats in E.coli become vulnerable after transcription. H-NS and its facilitated IS1E transposition can silence long triplet repeats transcription and preserve cell growth and survival. PMID:21151567

  13. 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.

  14. Simultaneous post-transcriptional gene silencing of two different chalcone synthase genes resulting in pure white flowers in the octoploid dahlia.

    PubMed

    Ohno, Sho; Hosokawa, Munetaka; Kojima, Misa; Kitamura, Yoshikuni; Hoshino, Atsushi; Tatsuzawa, Fumi; Doi, Motoaki; Yazawa, Susumu

    2011-11-01

    Garden dahlias (Dahlia variabilis) are autoallooctoploids with redundant genes producing wide color variations in flowers. There are no pure white dahlia cultivars, despite its long breeding history. However, the white areas of bicolor flower petals appear to be pure white. The objective of this experiment was to elucidate the mechanism by which the pure white color is expressed in the petals of some bicolor cultivars. A pigment analysis showed that no flavonoid derivatives were detected in the white areas of petals in a star-type cultivar 'Yuino' and the two seedling cultivars 'OriW1' and 'OriW2' borne from a red-white bicolor cultivar, 'Orihime', indicating that their white areas are pure white. Semi-quantitative RT-PCR showed that in the pure white areas, transcripts of two chalcone synthases (CHS), DvCHS1 and DvCHS2 which share 69% nucleotide similarity with each other, were barely detected. Premature mRNA of DvCHS1 and DvCHS2 were detected, indicating that these two CHS genes are silenced post-transcriptionally. RNA gel blot analysis revealed that small interfering RNAs (siRNAs) derived from CHSs were produced in these pure white areas. By high-throughput sequence analysis of small RNAs in the pure white areas with no mismatch acceptance, small RNAs were mapped to two alleles of DvCHS1 and two alleles of DvCHS2 expressed in 'Yuino' petals. Therefore, we concluded that simultaneous siRNA-mediated post-transcriptional gene silencing of redundant CHS genes results in the appearance of pure white color in dahlias.

  15. Protective effect of caffeine against high sugar-induced transcription of microRNAs and consequent gene silencing: A study using lenses of galactosemic mice

    PubMed Central

    Kovtun, Svitlana

    2013-01-01

    Purpose Previous studies have shown that caffeine prevents the formation of cataracts induced by a high-galactose diet and consequent oxidative stress. The objective of this study was to investigate if this protective effect is reflected in the attenuation of the transcription of microRNAs (miRNAs) known to induce apoptosis and cell death by gene silencing. Methods Young CD-1 mice were fed either a normal laboratory diet or a diet containing 25% galactose with or without 1% caffeine. One week later, the animals were euthanized, and the lenses isolated and promptly processed for RNA isolation and subsequent preparation of cDNAs by reverse transcriptase reaction. Mature miRNA (miR)-specific cDNAs were then quantified with PCR in a 96-well microRNA-specific cassette using an ABI7900HT PCR machine. Results As expected from previous studies, the lenses were positive for all 84 miRs corresponding to the miRNA probes present in the cassette wells. However, the levels of at least 19 miRs were significantly elevated in galactosemic lenses compared to those in the normal lenses. The majority are proapoptotic. Such elevation was inhibited by caffeine. This has been demonstrated for the first time. Conclusions Since aberrant elevation of miRNAs silences various genes and consequently deactivates protein translation, and since caffeine downregulates such aberration, the beneficial effect of caffeine could be attributed to its ability to suppress elevation of toxic miRs and consequent gene silencing. PMID:23441122

  16. [Sop proteins can cause transcriptional silencing of genes located close to the centromere sites of linear plasmid N15].

    PubMed

    Mardanov, A V; Lane, D; Ravin, N V

    2010-01-01

    Stable inheritance of bacterial chromosomes and low copy number plasmids is ensured by accurate partitioning of replicated molecules between the daughter cells at division. Partitioning of the prophage of the temperate bacteriophage N15, which exists as a linear plasmid molecule with covalently closed ends, depends on the sop locus, comprising genes sopA and sopB, as well as four centromere sites located in different regions of the N15 genome essential for replication and the control of lysogeny. We found that binding of SopB to the centromere can silence centromere-proximal promoters, presumably due to subsequent polymerizing of SopB along the DNA. Close to the IR4 centromere site we identified a promoter, P59, able to drive expression of phage late genes encoding the structural proteins of virion. We found that following binding to IR4 the N15 Sop proteins can cause repression of this promoter. The repression depends on SopB and became stronger in the presence of SopA. Sop-dependent silencing of centromere-proximal promoters control gene expression in phage N15, particularly preventing undesired expression of late genes in the N15 prophage. Thus, the phage N15 sop system not only ensures plasmid partitioning but is also involved in the genetic network controlling prophage replication and the maintenance of lysogeny.

  17. Silencing of molt-regulating transcription factor gene, CiHR3, affects growth and development of sugarcane stem borer, Chilo infuscatellus.

    PubMed

    Zhang, Yu-liang; Zhang, Shu-zhen; Kulye, Mahesh; Wu, Su-ran; Yu, Nai-tong; Wang, Jian-hua; Zeng, Hong-mei; Liu, Zhi-xin

    2012-01-01

    RNA interference (RNAi) is a technology for conducting functional genomic studies and a potential tool for crop protection against insect pests. Development of reliable methods for production and delivery of double-stranded RNA (dsRNA) is the major challenge for efficient pest control. In this study, Chilo infuscatellus Snellen (Crambidae: Lepidoptera) was fed with CiHR3 dsRNA expressed in bacteria or synthesized in vitro. The dsRNA ingested by C. infuscatellus successfully triggered silencing of the molt-regulating transcription factor CiHR3, an important gene for insect growth and development, and caused significant abnormalities and weight loss in insects within seven days of treatment. This study is an ideal example of feeding-based RNAi mediated by dsRNA expressed in bacteria or synthesized in vitro. The results also suggested that feeding-based RNA interference is a potential method for the management of C. infuscatellus.

  18. Silencing of Molt-Regulating Transcription Factor Gene, CiHR3, Affects Growth and Development of Sugarcane Stem Borer, Chilo infuscatellus

    PubMed Central

    Zhang, Yu-liang; Zhang, Shu-zhen; Kulye, Mahesh; Wu, Su-ran; Yu, Nai-tong; Wang, Jian-hua; Zeng, Hong-mei; Liu, Zhi-xin

    2012-01-01

    RNA interference (RNAi) is a technology for conducting functional genomic studies and a potential tool for crop protection against insect pests. Development of reliable methods for production and delivery of double-stranded RNA (dsRNA) is the major challenge for efficient pest control. In this study, Chilo infuscatellus Snellen (Crambidae: Lepidoptera) was fed with CiHR3 dsRNA expressed in bacteria or synthesized in vitro. The dsRNA ingested by C. infuscatellus successfully triggered silencing of the molt-regulating transcription factor CiHR3, an important gene for insect growth and development, and caused significant abnormalities and weight loss in insects within seven days of treatment. This study is an ideal example of feeding-based RNAi mediated by dsRNA expressed in bacteria or synthesized in vitro. The results also suggested that feeding-based RNA interference is a potential method for the management of C. infuscatellus. PMID:23427912

  19. Enhancer decommissioning by Snail1-induced competitive displacement of TCF7L2 and down-regulation of transcriptional activators results in EPHB2 silencing.

    PubMed

    Schnappauf, Oskar; Beyes, Sven; Dertmann, Annika; Freihen, Vivien; Frey, Patrick; Jägle, Sabine; Rose, Katja; Michoel, Tom; Grosschedl, Rudolf; Hecht, Andreas

    2016-11-01

    Transcriptional silencing is a major cause for the inactivation of tumor suppressor genes, however, the underlying mechanisms are only poorly understood. The EPHB2 gene encodes a receptor tyrosine kinase that controls epithelial cell migration and allocation in intestinal crypts. Through its ability to restrict cell spreading, EPHB2 functions as a tumor suppressor in colorectal cancer whose expression is frequently lost as tumors progress to the carcinoma stage. Previously we reported that EPHB2 expression depends on a transcriptional enhancer whose activity is diminished in EPHB2 non-expressing cells. Here we investigated the mechanisms that lead to EPHB2 enhancer inactivation. We show that expression of EPHB2 and SNAIL1 - an inducer of epithelial-mesenchymal transition (EMT) - is anti-correlated in colorectal cancer cell lines and tumors. In a cellular model of Snail1-induced EMT, we observe that features of active chromatin at the EPHB2 enhancer are diminished upon expression of murine Snail1. We identify the transcription factors FOXA1, MYB, CDX2 and TCF7L2 as EPHB2 enhancer factors and demonstrate that Snail1 indirectly inactivates the EPHB2 enhancer by downregulation of FOXA1 and MYB. In addition, Snail1 induces the expression of Lymphoid enhancer factor 1 (LEF1) which competitively displaces TCF7L2 from the EPHB2 enhancer. In contrast to TCF7L2, however, LEF1 appears to repress the EPHB2 enhancer. Our findings underscore the importance of transcriptional enhancers for gene regulation under physiological and pathological conditions and show that SNAIL1 employs a combinatorial mechanism to inactivate the EPHB2 enhancer based on activator deprivation and competitive displacement of transcription factors. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Mungbean yellow mosaic virus (MYMV) AC4 suppresses post-transcriptional gene silencing and an AC4 hairpin RNA gene reduces MYMV DNA accumulation in transgenic tobacco.

    PubMed

    Sunitha, Sukumaran; Shanmugapriya, Gnanasekaran; Balamani, Veluthambi; Veluthambi, Karuppannan

    2013-06-01

    Mungbean yellow mosaic virus (MYMV) is a legume-infecting geminivirus that causes yellow mosaic disease in blackgram, mungbean, soybean, Frenchbean and mothbean. AC4/C4, which is nested completely within the Rep gene, is less conserved among geminiviruses. Much less is known about its role in viral pathogenesis other than its known role in the suppression of host-mediated gene silencing. Transient expression of MYMV AC4 by agroinfiltration suppressed post-transcriptional gene silencing in Nicotiana benthamiana 16c expressing green fluorescence protein, at a level comparable to MYMV TrAP expression. AC4 full-length gene and an inverted repeat of AC4 (comprising the full-length AC4 sequence in sense and antisense orientations with an intervening intron) which makes a hairpin RNA (hpRNA) upon transcription were introduced into tobacco by Agrobacterium-mediated leaf disc transformation. Leaf discs of the transgenic plants were agroinoculated with partial dimers of MYMV and used to study the effect of the AC4-sense and AC4 hpRNA genes on MYMV DNA accumulation. Leaf discs of two transgenic plants that express the AC4-sense gene displayed an increase in MYMV DNA accumulation. Leaf discs of six transgenic plants containing the AC4 hpRNA gene accumulated small-interfering RNAs (siRNAs) specific to AC4, and upon agroinoculation with MYMV they exhibited a severe reduction in the accumulation of MYMV DNA. Thus, the MYMV AC4 hpRNA gene has emerged as a good candidate to engineer resistance against MYMV in susceptible plants.

  1. Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region

    NASA Technical Reports Server (NTRS)

    Mazumder, B.; Fox, P. L.

    1999-01-01

    Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine

  2. Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region

    NASA Technical Reports Server (NTRS)

    Mazumder, B.; Fox, P. L.

    1999-01-01

    Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine

  3. Neuron-restrictive Silencer Factor (NRSF) Represses Cocaine- and Amphetamine-regulated Transcript (CART) Transcription and Antagonizes cAMP-response Element-binding Protein Signaling through a Dual NRSE Mechanism*

    PubMed Central

    Zhang, Jing; Wang, Sihan; Yuan, Lin; Yang, Yinxiang; Zhang, Bowen; Liu, Qingbin; Chen, Lin; Yue, Wen; Li, Yanhua; Pei, Xuetao

    2012-01-01

    Cocaine- and amphetamine-regulated transcript (CART) peptide plays a pivotal role in neuroprotection against stroke-related brain injury. However, the regulatory mechanism on CART transcription, especially the repression mechanism, is not fully understood. Here, we show that the transcriptional repressor neuron-restrictive silencer elements (NRSF, also known as REST) represses CART expression through direct binding to two NRSF-binding elements (NRSEs) in the CART promoter and intron 1 (named pNRSE and iNRSE, respectively). EMSA show that NRSF binds to pNRSE and iNRSE directly in vitro. ChIP assays show that NRSF recruits differential co-repressor complexes including CoREST and HDAC1 to these NRSEs. The presence of both NRSEs is required for efficient repression of CART transcription as indicated by reporter gene assays. NRSF overexpression antagonizes forskolin-mediated up-regulation of CART mRNA and protein. Ischemia insult triggered by oxygen-glucose deprivation (OGD) enhances NRSF mRNA levels and then NRSF antagonizes the CREB signaling on CART activation, leading to augmented cell death. Depletion of NRSF in combination with forskolin treatment increases neuronal survival after ischemic insult. These findings reveal a novel dual NRSE mechanism by which NRSF represses CART expression and suggest that NRSF may serve as a therapeutic target for stroke treatment. PMID:23086924

  4. Epigenetic silencing in transgenic plants

    PubMed Central

    Rajeevkumar, Sarma; Anunanthini, Pushpanathan; Sathishkumar, Ramalingam

    2015-01-01

    Epigenetic silencing is a natural phenomenon in which the expression of genes is regulated through modifications of DNA, RNA, or histone proteins. It is a mechanism for defending host genomes against the effects of transposable elements and viral infection, and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was the discovery of silencing in transgenic tobacco plants due to the interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, that is, transcriptional gene silencing, which is associated with heavy methylation of promoter regions and blocks the transcription of transgenes, and post-transcriptional gene silencing (PTGS), the basic mechanism is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of major concern in the transgenic technologies used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes is required. The current status of epigenetic silencing in transgenic technology is discussed and summarized in this mini-review. PMID:26442010

  5. MiR-1254 suppresses HO-1 expression through seed region-dependent silencing and non-seed interaction with TFAP2A transcript to attenuate NSCLC growth

    PubMed Central

    Qi, Xinming; Chen, Jing; Wang, Yizheng; Gao, Lulu; Miao, Lingling; Ren, Jin

    2017-01-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs, which direct post-transcriptional gene silencing (PTGS) and function in a vast range of biological events including cancer development. Most miRNAs pair to the target sites through seed region near the 5’ end, leading to mRNA cleavage and/or translation repression. Here, we demonstrated a miRNA-induced dual regulation of heme oxygenase-1 (HO-1) via seed region and non-seed region, consequently inhibited tumor growth of NSCLC. We identified miR-1254 as a negative regulator inhibiting HO-1 translation by directly targeting HO-1 3’UTR via its seed region, and suppressing HO-1 transcription via non-seed region-dependent inhibition of transcriptional factor AP-2 alpha (TFAP2A), a transcriptional activator of HO-1. MiR-1254 induced cell apoptosis and cell cycle arrest in human non-small cell lung carcinoma (NSCLC) cells by inhibiting the expression of HO-1, consequently suppressed NSCLC cell growth. Consistently with the in vitro studies, mouse xenograft studies validated that miR-1254 suppressed NSCLC tumor growth in vivo. Moreover, we found that HO-1 expression was inversely correlated with miR-1254 level in human NSCLC tumor samples and cell lines. Overall, these findings identify the dual inhibition of HO-1 by miR-1254 as a novel functional mechanism of miRNA, which results in a more effective inhibition of oncogenic mRNA, and leads to a tumor suppressive effect. PMID:28749936

  6. MiR-1254 suppresses HO-1 expression through seed region-dependent silencing and non-seed interaction with TFAP2A transcript to attenuate NSCLC growth.

    PubMed

    Pu, Mengfan; Li, Chenggang; Qi, Xinming; Chen, Jing; Wang, Yizheng; Gao, Lulu; Miao, Lingling; Ren, Jin

    2017-07-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs, which direct post-transcriptional gene silencing (PTGS) and function in a vast range of biological events including cancer development. Most miRNAs pair to the target sites through seed region near the 5' end, leading to mRNA cleavage and/or translation repression. Here, we demonstrated a miRNA-induced dual regulation of heme oxygenase-1 (HO-1) via seed region and non-seed region, consequently inhibited tumor growth of NSCLC. We identified miR-1254 as a negative regulator inhibiting HO-1 translation by directly targeting HO-1 3'UTR via its seed region, and suppressing HO-1 transcription via non-seed region-dependent inhibition of transcriptional factor AP-2 alpha (TFAP2A), a transcriptional activator of HO-1. MiR-1254 induced cell apoptosis and cell cycle arrest in human non-small cell lung carcinoma (NSCLC) cells by inhibiting the expression of HO-1, consequently suppressed NSCLC cell growth. Consistently with the in vitro studies, mouse xenograft studies validated that miR-1254 suppressed NSCLC tumor growth in vivo. Moreover, we found that HO-1 expression was inversely correlated with miR-1254 level in human NSCLC tumor samples and cell lines. Overall, these findings identify the dual inhibition of HO-1 by miR-1254 as a novel functional mechanism of miRNA, which results in a more effective inhibition of oncogenic mRNA, and leads to a tumor suppressive effect.

  7. Dnmt3b recruitment through E2F6 transcriptional repressor mediates germ-line gene silencing in murine somatic tissues

    PubMed Central

    Velasco, Guillaume; Hubé, Florent; Rollin, Jérôme; Neuillet, Damien; Philippe, Cathy; Bouzinba-Segard, Haniaa; Galvani, Angélique; Viegas-Péquignot, Evani; Francastel, Claire

    2010-01-01

    Methylation of cytosine residues within the CpG dinucleotide in mammalian cells is an important mediator of gene expression, genome stability, X-chromosome inactivation, genomic imprinting, chromatin structure, and embryonic development. The majority of CpG sites in mammalian cells is methylated in a nonrandom fashion, raising the question of how DNA methylation is distributed along the genome. Here, we focused on the functions of DNA methyltransferase-3b (Dnmt3b), of which deregulated activity is linked to several human pathologies. We generated Dnmt3b hypomorphic mutant mice with reduced catalytic activity, which first revealed a deregulation of Hox genes expression, consistent with the observed homeotic transformations of the posterior axis. In addition, analysis of deregulated expression programs in Dnmt3b mutant embryos, using DNA microarrays, highlighted illegitimate activation of several germ-line genes in somatic tissues that appeared to be linked directly to their hypomethylation in mutant embryos. We provide evidence that these genes are direct targets of Dnmt3b. Moreover, the recruitment of Dnmt3b to their proximal promoter is dependant on the binding of the E2F6 transcriptional repressor, which emerges as a common hallmark in the promoters of genes found to be up-regulated as a consequence of impaired Dnmt3b activity. Therefore, our results unraveled a coordinated regulation of genes involved in meiosis, through E2F6-dependant methylation and transcriptional silencing in somatic tissues. PMID:20439742

  8. Transcriptional silencing of the Wnt-antagonist DKK1 by promoter methylation is associated with enhanced Wnt signaling in advanced multiple myeloma.

    PubMed

    Kocemba, Kinga A; Groen, Richard W J; van Andel, Harmen; Kersten, Marie José; Mahtouk, Karène; Spaargaren, Marcel; Pals, Steven T

    2012-01-01

    The Wnt/β-catenin pathway plays a crucial role in the pathogenesis of various human cancers. In multiple myeloma (MM), aberrant auto-and/or paracrine activation of canonical Wnt signaling promotes proliferation and dissemination, while overexpression of the Wnt inhibitor Dickkopf1 (DKK1) by MM cells contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/β-catenin mediated transcription, these findings suggest that DKK1 is part of a negative feedback loop in MM and may act as a tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is low or undetectable in a subset of patients with advanced MM as well as in MM cell lines. This absence of DKK1 is correlated with enhanced Wnt pathway activation, evidenced by nuclear accumulation of β-catenin, which in turn can be antagonized by restoring DKK1 expression. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling.

  9. Dnmt3b recruitment through E2F6 transcriptional repressor mediates germ-line gene silencing in murine somatic tissues.

    PubMed

    Velasco, Guillaume; Hubé, Florent; Rollin, Jérôme; Neuillet, Damien; Philippe, Cathy; Bouzinba-Segard, Haniaa; Galvani, Angélique; Viegas-Péquignot, Evani; Francastel, Claire

    2010-05-18

    Methylation of cytosine residues within the CpG dinucleotide in mammalian cells is an important mediator of gene expression, genome stability, X-chromosome inactivation, genomic imprinting, chromatin structure, and embryonic development. The majority of CpG sites in mammalian cells is methylated in a nonrandom fashion, raising the question of how DNA methylation is distributed along the genome. Here, we focused on the functions of DNA methyltransferase-3b (Dnmt3b), of which deregulated activity is linked to several human pathologies. We generated Dnmt3b hypomorphic mutant mice with reduced catalytic activity, which first revealed a deregulation of Hox genes expression, consistent with the observed homeotic transformations of the posterior axis. In addition, analysis of deregulated expression programs in Dnmt3b mutant embryos, using DNA microarrays, highlighted illegitimate activation of several germ-line genes in somatic tissues that appeared to be linked directly to their hypomethylation in mutant embryos. We provide evidence that these genes are direct targets of Dnmt3b. Moreover, the recruitment of Dnmt3b to their proximal promoter is dependant on the binding of the E2F6 transcriptional repressor, which emerges as a common hallmark in the promoters of genes found to be up-regulated as a consequence of impaired Dnmt3b activity. Therefore, our results unraveled a coordinated regulation of genes involved in meiosis, through E2F6-dependant methylation and transcriptional silencing in somatic tissues.

  10. Bacterial toxins induce sustained mRNA expression of the silencing transcription factor klf2 via inactivation of RhoA and Rhophilin 1.

    PubMed

    Dach, Kristina; Zovko, Josip; Hogardt, Michael; Koch, Isabel; van Erp, Katrin; Heesemann, Jürgen; Hoffmann, Reinhard

    2009-12-01

    Yersiniae bearing the Yersinia virulence plasmid pYV impact the transcriptome of J774A.1 macrophage-like cells in two distinct ways: (i) by suppressing, in a Yersinia outer protein P (YopP)-dependent manner, the induction of inflammatory response genes and (ii) by mRNA induction of the silencing transcription factor klf2. Here we show that klf2 induction by Yersinia enterocolitica occurs in several cell lines of macrophage and squamous and upper gastrointestinal epithelial origin as well as in bone marrow-derived dendritic cells. Several strains of Pseudomonas aeruginosa and Staphylococcus aureus are equally effective as Y. enterocolitica in inducing klf2 expression. Screening of mutant strains or incubation with recombinant toxins identified the rho-inactivating toxins YopT from Yersinia spp., ExoS from Pseudomonas aeruginosa, EDIN-B from Staphylococcus aureus, and C3bot from Clostridium botulinum as bacterial inducers of klf2 mRNA. klf2 mRNA induction by these toxins does not require de novo protein synthesis. Serum response factor or actin depolymerization does not seem to be involved in regulating klf2 expression in response to bacterial infection. Instead, short hairpin RNA-mediated inactivation of RhoA and its effector rhophilin 1 is sufficient to induce long-term klf2 expression. Thus, bacteria exploit the RhoA-rhophilin signaling cascade to mediate sustained expression of the immunosuppressive transcription factor klf2.

  11. Transcriptional silencing of ETS-1 abrogates epithelial-mesenchymal transition resulting in reduced motility of pancreatic cancer cells.

    PubMed

    Li, Chunyan; Wang, Zhonghan; Chen, Yan; Zhou, Min; Zhang, Haijun; Chen, Rong; Shi, Fangfang; Wang, Cailian; Rui, Zongdao

    2015-02-01

    v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS-1) plays crucial roles in a spectrum of malignancies. ETS-1 has gained attention in cancer research for its importance in cell migration, invasion and proliferation. In the present study, we focused on the effect of ETS-1 on epithelial-mesenchymal transition (EMT), which is characterized by reduced E-cadherin expression and increased N-cadherin expression. We found that ETS-1 mRNA expression was positively correlated with N-cadherin and negatively correlated with E-cadherin mRNA expression in five pancreatic cancer cell lines. To elucidate the functionality of ETS-1 on EMT in pancreatic cancer cells, we constructed a green fluorescent protein (GFP)-expressing plasmid carrying ETS-1 short hairpin RNA (shRNA), and transfected Panc-1 cells with the plasmid. We detected reduced N-cadherin and vascular endothelial growth factor yet higher E-cadherin expression in the ETS-1-silenced cells compared with the control group. In addition, we observed reduced cell migration and increased adhesion in these cells. Our data showed that ETS-1 actively functioned as a regulator of EMT in Panc-1 cells, and provide additional evidence supporting a fundamental role for ETS-1 in metastatic pancreatic cancer cells. These results suggest that analysis of ETS-1 expression levels may provide an avenue for evaluating prognosis in pancreatic cancer.

  12. The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion.

    PubMed

    Masroori, Nasser; Merindol, Natacha; Berthoux, Lionel

    2016-03-22

    The promyelocytic leukemia (PML) protein, a type I interferon (IFN-I)-induced gene product and a member of the tripartite motif (TRIM) family, modulates the transcriptional activity of viruses belonging to various families. Whether PML has an impact on the replication of HIV-1 has not been fully addressed, but recent studies point to its possible involvement in the restriction of HIV-1 in human cells and in the maintenance of transcriptional latency in human cell lines in which HIV-1 is constitutively repressed. We investigated further the restriction of HIV-1 and a related lentivirus, SIVmac, by PML in murine cells and in a lymphocytic human cell line. In particular, we studied the relevance of PML to IFN-I-mediated inhibition and the role of individual human isoforms. We demonstrate that both human PML (hPML) and murine PML (mPML) inhibit the early post-entry stages of the replication of HIV-1 and a related lentivirus, SIVmac. In addition, HIV-1 was transcriptionally silenced by mPML and by hPML isoforms I, II, IV and VI in MEFs. This PML-mediated transcriptional repression was attenuated in presence of the histone deacetylase inhibitor SAHA. In contrast, depletion of PML had no effect on HIV-1 gene expression in a human T cell line. PML was found to contribute to the inhibition of HIV-1 by IFN-I. Specifically, IFN-α and IFN-β treatments of MEFs enhanced the PML-dependent inhibition of HIV-1 early replication stages. We show that PML can inhibit HIV-1 and other lentiviruses as part of the IFN-I-mediated response. The restriction takes place at two distinct steps, i.e. reverse transcription and transcription, and in an isoform-specific, cellular context-specific fashion. Our results support a model in which PML activates innate immune antilentiviral effectors. These data are relevant to the development of latency reversal-inducing pharmacological agents, since PML was previously proposed as a pharmacological target for such inhibitors. This study also has

  13. Transcript expression bias of phosphatidylethanolamine binding protein gene in bumblebee, Bombus lantschouensis (Hymenoptera: Apidae).

    PubMed

    Dong, Jie; Han, Lei; Wang, Ye; Huang, Jiaxing; Wu, Jie

    2017-09-05

    The phosphatidylethanolamine-binding protein (PEBP) family is a highly conserved group of proteins found in a wide range of organism. It plays an important role in innate immunity of insects. Little is known on the expression characteristic and function of PEBP in bees. In the current study, we cloned the pebp gene and investigated its expression profiles at different developmental stages and reproductive status from bumblebee, Bombus lantschouensis (Vogt), which is one of the most abundant pollinators for wild plants and crops in Northern China. Two transcripts (PEBPX1 and PEBPX2) of the pebp gene were cloned for the first time. The transcript PEBPX2 lacked a signal peptide sequence compared to PEBPX1. The full-length cDNA of these two PEBP transcripts is 1005bp and 915bp, with an open reading frame of 627bp and 549bp, respectively. Transcript PEBPX2 was one order of magnitude more expressed than transcript PEBPX1 at most of the developmental stages and different reproductive status (egg-laying versus non- egg-laying females). Both of the PEBP transcripts were highly expressed in brown-eyed with light and dark pigmented cuticle pupae stages. Quantitative PCR and Western Blot demonstrated that PEBP was significantly up-regulated in egg-laying females. In summary, we suggest that levels of these two PEBPs could be related to the regulation of reproduction in bumblebees. In addition, both transcripts likely play an important role in the metamorphosis developmental stage of bumblebee pupae. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Inhibiting cell migration and cell invasion by silencing the transcription factor ETS-1 in human bladder cancer.

    PubMed

    Liu, Li; Liu, Yuchen; Zhang, Xintao; Chen, Mingwei; Wu, Hanwei; Lin, Muqi; Zhan, Yonghao; Zhuang, Chengle; Lin, Junhao; Li, Jianfa; Xu, Wen; Fu, Xing; Zhang, Qiaoxia; Sun, Xiaojuan; Zhao, Guoping; Huang, Weiren

    2016-05-03

    As one of the members of the ETS gene family, the transcription factor v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS-1) plays key role in the regulation of physiological processes in normal cells and tumors. In this study, we aimed to investigate the relationship between the transcription factor ETS-1 and malignant phenotypes of bladder cancer. We demonstrated that ETS-1 was up-regulated in human bladder cancer tissue compared to paired normal bladder tissue. In order to evaluate the functional role of ETS-1 in human bladder cancer, vectors expressing ETS-1 shRNA and ETS-1 protein were constructed in vitro and transfected into the human bladder cancer T24 and 5637 cells. Our results showed that the transcription factor ETS-1 could promote cell migration and cell invasion in human bladder cancer, without affecting cell proliferation and apoptosis. In conclusion, ETS-1 plays oncogenic roles through inducing cell migration and invasion in human bladder cancer, and it can be used as a therapeutic target for treating human bladder cancer.

  15. Epigenetic silencing of JMJD5 promotes the proliferation of hepatocellular carcinoma cells by down-regulating the transcription of CDKN1A

    PubMed Central

    Fang, Jia-Zhu; Wu, Chong-Chao; Huang, Li-Yu; Wang, Lan; Han, Ze-Guang

    2016-01-01

    Proteins that contain jumonji C (JmjC) domains have recently been identified as major contributors to various malignant human cancers through epigenetic remodeling. However, the roles of these family members in the pathogenesis of hepatocellular carcinoma (HCC) are obscure. By mining public databases, we found that the HCC patients with lower JmjC domain-containing protein 5 (JMJD5) expression exhibited shorter survival time. We then confirmed that JMJD5 expression was indeed decreased in HCC specimens, which was caused by the altered epigenetic histone modifications, the decreased H3K9ac, H3K27ac and H3K4me2/3 together with the increased trimethylation of H3K27 and H3K9 on the JMJD5 promoter. Functional experiments revealed that JMJD5 knockdown promoted HCC cell proliferation and in vivo tumorigenicity by accelerating the G1/S transition of the cell cycle; in contrast, ectopic JMJD5 expression had the opposite effects. At molecular mechanism, we found that, in HCC cell lines including TP53-null Hep3B, JMJD5 knockdown led to the down-regulation of CDKN1A and ectopic expression of JMJD5 not only increased but also rescued CDKN1A transcription. Moreover, CDKN1A knockdown could abrogate the effect of JMJD5 knockdown or overexpression on cell proliferation, suggesting that JMJD5 inhibits HCC cell proliferation mainly by activating CDKN1A expression. We further revealed that JMJD5 directly enhances CDKN1A transcription by binding to CDKN1A's promoter independent of H3K36me2 demethylase activity. In short, we first prove that JMJD5 is a tumor suppressor gene in HCC pathogenesis, and the epigenetic silencing of JMJD5 promotes HCC cell proliferation by directly down-regulating CDKN1A transcription. PMID:26760772

  16. Deconstructing Phonetic Transcription: Covert Contrast, Perceptual Bias, and an Extraterrestrial View of "Vox Humana"

    ERIC Educational Resources Information Center

    Munson, Benjamin; Edwards, Jan; Schellinger, Sarah K.; Beckman, Mary E.; Meyer, Marie K.

    2010-01-01

    This article honours Adele Miccio's life work by reflecting on the utility of phonetic transcription. The first section reviews the literature on cases where children whose speech appears to neutralize a contrast in the adult language are found on closer examination to produce a contrast ("covert contrast"). This study presents evidence…

  17. Deconstructing Phonetic Transcription: Covert Contrast, Perceptual Bias, and an Extraterrestrial View of "Vox Humana"

    ERIC Educational Resources Information Center

    Munson, Benjamin; Edwards, Jan; Schellinger, Sarah K.; Beckman, Mary E.; Meyer, Marie K.

    2010-01-01

    This article honours Adele Miccio's life work by reflecting on the utility of phonetic transcription. The first section reviews the literature on cases where children whose speech appears to neutralize a contrast in the adult language are found on closer examination to produce a contrast ("covert contrast"). This study presents evidence…

  18. MYC suppresses cancer metastasis by direct transcriptional silencing of αv and β3 integrin subunits.

    PubMed

    Liu, Hong; Radisky, Derek C; Yang, Dun; Xu, Ren; Radisky, Evette S; Bissell, Mina J; Bishop, J Michael

    2012-05-13

    Overexpression of MYC transforms cells in culture, elicits malignant tumours in experimental animals and is found in many human tumours. We now report the paradoxical finding that this powerful oncogene can also act as a suppressor of cell motility, invasiveness and metastasis. Overexpression of MYC stimulated proliferation of breast cancer cells both in culture and in vivo as expected, but inhibited motility and invasiveness in culture, and lung and liver metastases in xenografted tumours. We show further that MYC represses transcription of both subunits of αvβ3 integrin, and that exogenous expression of β3 integrin in human breast cancer cells that do not express this integrin rescues invasiveness and migration when MYC is downregulated. These data uncover an unexpected function of MYC, provide an explanation for the hitherto puzzling literature on the relationship between MYC and metastasis, and reveal a variable that could influence the development of therapies that target MYC.

  19. Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana.

    PubMed

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2014-06-01

    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  20. Transgenic resistance in potato plants expressing potato leaf roll virus (PLRV) replicase gene sequences is RNA-mediated and suggests the involvement of post-transcriptional gene silencing.

    PubMed

    Vazquez Rovere, C; Asurmendi, S; Hopp, H E

    2001-07-01

    Genetically engineered expression of replicase encoding sequences has been proposed as an efficient system to confer protection against virus diseases by eliciting protection mechanisms in the plant. Potato leaf-roll was one of the first diseases for which this kind of protection was engineered in potato plants. However, details of the protecting mechanism were not reported, so far. The ORF2b of an Argentinean strain of PLRV was cloned and sequenced finding 94% and 97% of homology with Australian and Dutch strains, respectively. To elucidate the mechanism of protection against PLRV infection, three versions of ORF2b (non-translatable sense, translatable sense with an engineered ATG and antisense) were constructed under the control of the 35S CaMV promoter and the nos terminator and introduced in potato plants (cv. Kennebec) by Agrobacterium tumefaciens-mediated transformation. Grafting infection experiments showed that resistant transgenic plants could be obtained with any of the constructs, suggesting that the mechanism of protection is independent of the expression of protein and is RNA mediated. Field trial infection confirmed that resistant transgenic events were obtained. Biolistic transient transformation experiments of leaves derived from transgenic plants using a gene coding for the fusion protein GUS-ORF2b, followed by scoring of the number of GUS expressing leaf spots, supported that the protection is mediated by a post-transcriptional gene silencing mechanism.

  1. UV-C-Induced alleviation of transcriptional gene silencing through plant-plant communication: Key roles of jasmonic acid and salicylic acid pathways.

    PubMed

    Xu, Wei; Wang, Ting; Xu, Shaoxin; Li, Fanghua; Deng, Chenguang; Wu, Lijun; Wu, Yuejin; Bian, Po

    2016-08-01

    Plant stress responses at the epigenetic level are expected to allow more permanent changes of gene expression and potentially long-term adaptation. While it has been reported that plants subjected to adverse environments initiate various stress responses in their neighboring plants, little is known regarding epigenetic responses to external stresses mediated by plant-plant communication. In this study, we show that DNA repetitive elements of Arabidopsis thaliana, whose expression is inhibited epigenetically by transcriptional gene silencing (TGS) mechanism, are activated by UV-C irradiation through airborne plant-plant and plant-plant-plant communications, accompanied by DNA demethylation at CHH sites. Moreover, the TGS is alleviated by direct treatments with exogenous methyl jasmonate (MeJA) and methyl salicylate (MeSA). Further, the plant-plant and plant-plant-plant communications are blocked by mutations in the biosynthesis or signaling of jasmonic acid (JA) or salicylic acid (SA), indicating that JA and SA pathways are involved in the interplant communication for epigenetic responses. For the plant-plant-plant communication, stress cues are relayed to the last set of receiver plants by promoting the production of JA and SA signals in relaying plants, which exhibit upregulated expression of genes for JA and SA biosynthesis and enhanced emanation of MeJA and MeSA. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Functions of the Hha and YdgT Proteins in Transcriptional Silencing by the Nucleoid Proteins, H-NS and StpA, in Escherichia coli

    PubMed Central

    Ueda, Takeshi; Takahashi, Hiroki; Uyar, Ebru; Ishikawa, Shu; Ogasawara, Naotake; Oshima, Taku

    2013-01-01

    The Hha and YdgT proteins are suggested to modulate the expression of horizontally acquired genes by interacting with H-NS and StpA, which play central roles in the transcriptional silencing of such genes. However, it is also possible that Hha/YdgT repress gene expression independently of H-NS/StpA, as we have not fully understood the molecular mechanism through which Hha/YdgT modulate H-NS/StpA activity. To gain further insight into the basic functions of Hha/YdgT, we analysed the impact of hha/ydgT double inactivation on the transcriptome profile of Escherichia coli K-12, and compared the effects with that of hns/stpA double inactivation. In addition, we examined the effects of hha/ydgT inactivation on the chromosomal binding of H-NS, and conversely the effects of hns/stpA inactivation on the chromosomal binding of Hha. Our results demonstrated that the chromosomal binding of Hha requires H-NS/StpA, and is necessary for the repression of a subset of genes in the H-NS/StpA regulon. Furthermore, the distribution of H-NS binding around Hha/YdgT-dependent and -independent genes suggests that Hha/YdgT proteins modulate formation of the H-NS/StpA-DNA complex. PMID:23543115

  3. Feminization of Male Mouse Liver by Persistent Growth Hormone Stimulation: Activation of Sex-Biased Transcriptional Networks and Dynamic Changes in Chromatin States

    PubMed Central

    Lau-Corona, Dana; Suvorov, Alexander

    2017-01-01

    ABSTRACT Sex-dependent pituitary growth hormone (GH) secretory profiles—pulsatile in males and persistent in females—regulate the sex-biased, STAT5-dependent expression of hundreds of genes in mouse liver, imparting sex differences in hepatic drug/lipid metabolism and disease risk. Here, we examine transcriptional and epigenetic changes induced by continuous GH infusion (cGH) in male mice, which rapidly feminizes the temporal profile of liver STAT5 activity. cGH repressed 86% of male-biased genes and induced 68% of female-biased genes within 4 days; however, several highly female-specific genes showed weak or no feminization, even after 14 days of cGH treatment. Female-biased genes already in an active chromatin state in male liver generally showed early cGH responses; genes in an inactive chromatin state often responded late. Early cGH-responsive genes included those encoding two GH/STAT5-regulated transcriptional repressors: male-biased BCL6, which was repressed, and female-specific CUX2, which was induced. Male-biased genes activated by STAT5 and/or repressed by CUX2 were enriched for early cGH repression. Female-biased BCL6 targets were enriched for early cGH derepression. Changes in sex-specific chromatin accessibility and histone modifications accompanied these cGH-induced sex-biased gene expression changes. Thus, the temporal, sex-biased gene responses to persistent GH stimulation are dictated by GH/STAT5-regulated transcription factors arranged in a hierarchical network and by the dynamics of changes in sex-biased epigenetic states. PMID:28694329

  4. Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing.

    PubMed

    Schludi, Martin H; May, Stephanie; Grässer, Friedrich A; Rentzsch, Kristin; Kremmer, Elisabeth; Küpper, Clemens; Klopstock, Thomas; Arzberger, Thomas; Edbauer, Dieter

    2015-10-01

    A massive expansion of a GGGGCC repeat upstream of the C9orf72 coding region is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. Despite its intronic localization and lack of a canonical start codon, both strands are translated into aggregating dipeptide repeat (DPR) proteins: poly-GA, poly-GP, poly-GR, poly-PR and poly-PA. To address conflicting findings on the predominant toxicity of the different DPR species in model systems, we compared the expression pattern of the DPR proteins in rat primary neurons and postmortem brain and spinal cord of C9orf72 mutation patients. Only poly-GA overexpression closely mimicked the p62-positive neuronal cytoplasmic inclusions commonly observed for all DPR proteins in patients. In contrast, overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions. In patients, most of the less common neuronal intranuclear DPR inclusions were para-nucleolar and p62 positive. Neuronal nucleoli in C9orf72 cases showed normal size and morphology regardless of the presence of poly-GR and poly-PR inclusions arguing against widespread nucleolar stress, reported in cellular models. Colocalization of para-nucleolar DPR inclusions with heterochromatin and a marker of transcriptional repression (H3K9me2) indicates a link to gene transcription. In contrast, we detected numerous intranuclear DPR inclusions not associated with nucleolar structures in ependymal and subependymal cells. In patients, neuronal inclusions of poly-GR, poly-GP and the poly-GA interacting protein Unc119 were less abundant than poly-GA inclusions, but showed similar regional and subcellular distribution. Regardless of neurodegeneration, all inclusions were most abundant in neocortex, hippocampus and thalamus, with few inclusions in brain stem and spinal cord. In the granular cell layer of the cerebellum, poly-GA and Unc119 inclusions were significantly more abundant in cases with FTLD than in cases with MND and FTLD/MND. Poly

  5. Reversible male sterility in eggplant (Solanum melongena L.) by artificial microRNA-mediated silencing of general transcription factor genes.

    PubMed

    Toppino, Laura; Kooiker, Maarten; Lindner, Matias; Dreni, Ludovico; Rotino, Giuseppe L; Kater, Martin M

    2011-08-01

    Since decades, plant male sterility is considered a powerful tool for biological containment to minimize unwanted self-pollination for hybrid seed production. Furthermore, prevention of pollen dispersal also answers to concerns regarding transgene flow via pollen from Genetically Modified (GM) crops to traditional crop fields or wild relatives. We induced male sterility by suppressing endogenous general transcription factor genes, TAFs, using anther-specific promoters combined with artificial microRNA (amiRNA) technology (Schwab et al., 2006). The system was made reversible by the ethanol inducible expression of an amiRNA-insensitive form of the target gene. We provide proof of concept in eggplant, a cultivated crop belonging to the Solanaceae family that includes many important food crops. The transgenic eggplants that we generated are completely male sterile and fertility can be fully restored by short treatments with ethanol, confirming the efficiency but also the reliability of the system in view of open field cultivation. By combining this system with induced parthenocarpy (Rotino et al., 1997), we provide a novel example of complete transgene containment in eggplant, which enables biological mitigation measures for the benefit of coexistence or biosafety purposes for GM crop cultivation. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology and Blackwell Publishing Ltd.

  6. Methylation of tumour suppressor gene promoters in the presence and absence of transcriptional silencing in high hyperdiploid acute lymphoblastic leukaemia.

    PubMed

    Paulsson, Kajsa; An, Qian; Moorman, Anthony V; Parker, Helen; Molloy, Gael; Davies, Teresa; Griffiths, Mike; Ross, Fiona M; Irving, Julie; Harrison, Christine J; Young, Bryan D; Strefford, Jon C

    2009-03-01

    Promoter methylation is a common phenomenon in tumours, including haematological malignancies. In the present study, we investigated 36 cases of high hyperdiploid (>50 chromosomes) acute lymphoblastic leukaemia (ALL) with methylation-specific multiplex ligase-dependent probe amplification to determine the extent of aberrant methylation in this subgroup. The analysis, which comprised the promoters of 35 known tumour suppressor genes, showed that 16 genes displayed abnormal methylation in at least one case each. The highest number of methylated gene promoters seen in a single case was thirteen, with all but one case displaying methylation for at least one gene. The most common targets were ESR1 (29/36 cases; 81%), CADM1 (IGSF4, TSLC1; 25/36 cases; 69%), FHIT (24/36 cases; 67%) and RARB (22/36 cases; 61%). Interestingly, quantitative reverse transcription-polymerase chain reaction showed that although methylation of the CADM1 and RARB promoters resulted in the expected pattern of downregulation of the respective genes, no difference could be detected in FHIT expression between methylation-positive and -negative cases. Furthermore, TIMP3 was not expressed regardless of methylation status, showing that aberrant methylation does not always lead to gene expression changes. Taken together, our findings suggest that aberrant methylation of tumour suppressor gene promoters is a common phenomenon in high hyperdiploid ALL.

  7. Deconstructing Phonetic Transcription: Covert Contrast, Perceptual Bias, and an Extraterrestrial View of Vox Humana

    PubMed Central

    Munson, Benjamin; Edwards, Jan; Schellinger, Sarah; Beckman, Mary E.; Meyer, Marie K.

    2010-01-01

    This article honours Adele Miccio's life work by reflecting on the utility of phonetic transcription. The first section reviews the literature on cases where children whose speech appears to neutralize a contrast in the adult language are found on closer examination to produce a contrast (covert contrast). We present evidence from a new series of perception studies that covert contrast may be far more prevalent in children's speech than existing studies would suggest. The second section presents the results of a new study designed to examine whether naïve listeners' perception of children's /s/ and /θ/ productions can be changed experimentally when they are led to believe that the children who produced the sounds were older or younger. Here, it is shown that, under the right circumstances, adults report more tokens of /θ/ to be accurate productions of /s/ when they believe a talker to be an older child than when they believe the talker to be younger. This finding suggests that auditory information alone cannot be the sole basis for judging the accuracy of a sound. The final section presents recommendations for supplementing phonetic transcription with other measures, to gain a fuller picture of children's production abilities. PMID:20345255

  8. Deconstructing phonetic transcription: covert contrast, perceptual bias, and an extraterrestrial view of Vox Humana.

    PubMed

    Munson, Benjamin; Edwards, Jan; Schellinger, Sarah K; Beckman, Mary E; Meyer, Marie K

    2010-01-01

    This article honours Adele Miccio's life work by reflecting on the utility of phonetic transcription. The first section reviews the literature on cases where children whose speech appears to neutralize a contrast in the adult language are found on closer examination to produce a contrast (covert contrast). This study presents evidence from a new series of perception studies that covert contrast may be far more prevalent in children's speech than existing studies would suggest. The second section presents the results of a new study designed to examine whether naïve listeners' perception of children's /s/ and /theta/ productions can be changed experimentally when they are led to believe that the children who produced the sounds were older or younger. Here, it is shown that, under the right circumstances, adults report more tokens of /theta/ to be accurate productions of /s/ when they believe a talker to be an older child than when they believe the talker to be younger. This finding suggests that auditory information alone cannot be the sole basis for judging the accuracy of a sound. The final section presents recommendations for supplementing phonetic transcription with other measures, to gain a fuller picture of children's production abilities.

  9. Synthetic biology challenges long-held hypotheses in translation, codon bias and transcription.

    PubMed

    Forster, Anthony C

    2012-07-01

    Synthetic biology is a powerful experimental approach, not only for developing new biotechnology applications, but also for testing hypotheses in basic biological science. Here, examples from our research using the best model system, Escherichia coli, are reviewed. New evidence drawn from synthetic biology has overturned several long-standing hypotheses regarding the mechanisms of transcription and translation: (i) all native aminoacyl-tRNAs are not equally efficient in translation at equivalent concentrations; (ii) accommodation is not always rate limiting in translation, and may not be for any aminoacyl-tRNA; (iii) proline is the only N-alkyl-amino acid in the genetic code not because of special suitability for protein structure, but because of its comparatively high nucleophilicity; (iv) the usages of most sense codons in E. coli do not correlate with cognate tRNA abundances and (v) class II transcriptional pausing and termination by T7 RNA polymerase cannot be assumed to occur in vivo based on in vitro data. Implications of these conclusions for the biotechnology field are discussed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila

    PubMed Central

    2010-01-01

    Background In male Drosophila melanogaster, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator. Results MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity in vitro and UAS-DsRed activation in Drosophila. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-lacZ and UAS-arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS-arm-lacZ expression increased by about 2-fold in both sexes. Conclusions Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal

  11. Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila.

    PubMed

    Schiemann, Anja H; Li, Fang; Weake, Vikki M; Belikoff, Esther J; Klemmer, Kent C; Moore, Stanley A; Scott, Maxwell J

    2010-11-09

    In male Drosophila melanogaster, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator. MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity in vitro and UAS-DsRed activation in Drosophila. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-lacZ and UAS-arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS-arm-lacZ expression increased by about 2-fold in both sexes. Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in

  12. Suppression of methylation-mediated transcriptional gene silencing by βC1-SAHH protein interaction during geminivirus-betasatellite infection.

    PubMed

    Yang, Xiuling; Xie, Yan; Raja, Priya; Li, Sizhun; Wolf, Jamie N; Shen, Qingtang; Bisaro, David M; Zhou, Xueping

    2011-10-01

    DNA methylation is a fundamental epigenetic modification that regulates gene expression and represses endogenous transposons and invading DNA viruses. As a counter-defense, the geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a betasatellite called DNA β. This study presents evidence that suppression of methylation-mediated TGS by the sole betasatellite-encoded protein, βC1, is crucial to the association of Tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB). We show that TYLCCNB complements Beet curly top virus (BCTV) L2⁻ mutants deficient for methylation inhibition and TGS suppression, and that cytosine methylation levels in BCTV and TYLCCNV genomes, as well as the host genome, are substantially reduced by TYLCCNB or βC1 expression. We also demonstrate that while TYLCCNB or βC1 expression can reverse TGS, TYLCCNV by itself is ineffective. Thus its AC2/AL2 protein, known to have suppression activity in other geminiviruses, is likely a natural mutant in this respect. A yeast two-hybrid screen of candidate proteins, followed by bimolecular fluorescence complementation analysis, revealed that βC1 interacts with S-adenosyl homocysteine hydrolase (SAHH), a methyl cycle enzyme required for TGS. We further demonstrate that βC1 protein inhibits SAHH activity in vitro. That βC1 and other geminivirus proteins target the methyl cycle suggests that limiting its product, S-adenosyl methionine, may be a common viral strategy for methylation interference. We propose that inhibition of methylation and TGS by βC1 stabilizes geminivirus/betasatellite complexes.

  13. Suppression of Methylation-Mediated Transcriptional Gene Silencing by βC1-SAHH Protein Interaction during Geminivirus-Betasatellite Infection

    PubMed Central

    Raja, Priya; Li, Sizhun; Wolf, Jamie N.; Shen, Qingtang; Bisaro, David M.; Zhou, Xueping

    2011-01-01

    DNA methylation is a fundamental epigenetic modification that regulates gene expression and represses endogenous transposons and invading DNA viruses. As a counter-defense, the geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a betasatellite called DNA β. This study presents evidence that suppression of methylation-mediated TGS by the sole betasatellite-encoded protein, βC1, is crucial to the association of Tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB). We show that TYLCCNB complements Beet curly top virus (BCTV) L2- mutants deficient for methylation inhibition and TGS suppression, and that cytosine methylation levels in BCTV and TYLCCNV genomes, as well as the host genome, are substantially reduced by TYLCCNB or βC1 expression. We also demonstrate that while TYLCCNB or βC1 expression can reverse TGS, TYLCCNV by itself is ineffective. Thus its AC2/AL2 protein, known to have suppression activity in other geminiviruses, is likely a natural mutant in this respect. A yeast two-hybrid screen of candidate proteins, followed by bimolecular fluorescence complementation analysis, revealed that βC1 interacts with S-adenosyl homocysteine hydrolase (SAHH), a methyl cycle enzyme required for TGS. We further demonstrate that βC1 protein inhibits SAHH activity in vitro. That βC1 and other geminivirus proteins target the methyl cycle suggests that limiting its product, S-adenosyl methionine, may be a common viral strategy for methylation interference. We propose that inhibition of methylation and TGS by βC1 stabilizes geminivirus/betasatellite complexes. PMID:22028660

  14. RNAi-mediated silencing of prohormone convertase (PC) 5/6 expression leads to impairment in processing of cocaine- and amphetamine-regulated transcript (CART) precursor

    PubMed Central

    Stein, Jeffrey; Shah, Rohan; Steiner, Donald F.; Dey, Arunangsu

    2006-01-01

    Understanding the functions of the widely expressed PCs (prohormone/proprotein convertases), including PC5/6, furin and PACE4 (paired basic amino acid cleaving enzyme 4), in animal models is difficult since individual knockouts of these PCs in mice exhibit early embryonic lethality. To investigate the roles of PC5/6 in processing pro-CART (pro-cocaine- and amphetamine-regulated transcript), an important anorexigenic peptide precursor, we have generated GH3 cells silenced for PC5/6 expression by RNAi (RNA interference). We show, following transient knockdown of PC5/6 in these neuroendocrine cells, that generation of the two bioactive forms, CART I (amino acids 42–89/55–102) and CART II (amino acids 49–89/62–102), from pro-CART is impaired due to a lack particularly of the A isoform of PC5/6. The results indicate that PC5/6A shares specificities primarily with PC2 (PC5/6A

  15. Functional Diversity of Silencers in Budding Yeasts

    PubMed Central

    Sjöstrand, Jimmy O. O.; Kegel, Andreas; Åström, Stefan U.

    2002-01-01

    We studied the silencing of the cryptic mating-type loci HMLα and HMRa in the budding yeast Kluyveromyces lactis. A 102-bp minimal silencer fragment was defined that was both necessary and sufficient for silencing of HMLα. Mutagenesis of the silencer revealed three distinct regions (A, B, and C) that were important for silencing. Recombinant K. lactis ribosomal DNA enhancer binding protein 1 (Reb1p) could bind the silencer in vitro, and point mutations in the B box abolished both Reb1p binding and silencer function. Furthermore, strains carrying temperature-sensitive alleles of the REB1 gene derepressed the transcription of the HMLα1 gene at the nonpermissive temperature. A functional silencer element from the K. lactis cryptic HMRa locus was also identified, which contained both Reb1p binding sites and A boxes, strongly suggesting a general role for these sequences in K. lactis silencing. Our data indicate that different proteins bind to Kluyveromyces silencers than to Saccharomyces silencers. We suggest that the evolution of silencers is rapid in budding yeasts and discuss the similarities and differences between silencers in Saccharomyces and Kluyveromyces. PMID:12456003

  16. Cell-Specific mRNA Profiling of the Caenorhabditis elegans Somatic Gonadal Precursor Cells Identifies Suites of Sex-Biased and Gonad-Enriched Transcripts.

    PubMed

    Kroetz, Mary B; Zarkower, David

    2015-10-23

    The Caenorhabditis elegans somatic gonad differs greatly between the two sexes in its pattern of cell divisions, migration, and differentiation. Despite decades of study, the genetic pathways directing early gonadal development and establishing sexual dimorphism in the gonad remain largely unknown. To help define the genetic networks that regulate gonadal development, we employed cell-specific RNA-seq. We identified transcripts present in the somatic gonadal precursor cells and their daughter cells of each sex at the onset of sexual differentiation. We identified several hundred gonad-enriched transcripts, including the majority of known regulators of early gonadal development, and transgenic reporter analysis confirmed the effectiveness of this approach. Before the division of the somatic gonad precursors, few sex-biased gonadal transcripts were detectable; less than 6 hr later, after their division, we identified more than 250 sex-biased transcripts, of which about a third were enriched in the somatic gonad compared to the whole animal. This indicates that a robust sex-biased developmental program, some of it gonad-specific, initiates in the somatic gonadal precursor cells around the time of their first division. About 10% of male-biased transcripts had orthologs with male-biased expression in the early mouse gonad, suggesting possible conservation of gonad sex differentiation. Cell-specific analysis also identified approximately 70 previously unannotated mRNA isoforms that are enriched in the somatic gonad. Our data illustrate the power of cell-specific transcriptome analysis and suggest that early sex differentiation in the gonad is controlled by a relatively small suite of differentially expressed genes, even after dimorphism has become apparent.

  17. In vitro transcription activities of Pol IV, Pol V and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing

    SciTech Connect

    Haag, Jeremy R.; Ream, Thomas S.; Marasco, Michelle; Nicora, Carrie D.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2012-12-14

    In Arabidopsis, RNA-dependent DNA methylation and transcriptional silencing involves three nuclear RNA polymerases that are biochemically undefined: the presumptive DNA-dependent RNA polymerases, Pol IV and Pol V and the putative RNA-dependent RNA polymerase, RDR2. Here, we demonstrate their RNA polymerase activities in vitro. Unlike Pol II, Pols IV and V require an RNA primer, are insensitive to alpha-amanitin and differ in their ability to displace non-template DNA during transcription. Biogenesis of 24 nt small interfering RNAs (siRNAs) requires both Pol IV and RDR2, which physically associate in vivo. Pol IV does not require RDR2 for activity, but RDR2 is nonfunctional in the absence of associated Pol IV, suggesting that their coupling explains the channeling of Pol IV transcripts into double-stranded RNAs that are then diced into 24 nt siRNAs.

  18. The Fission Yeast Ubiquitin-Conjugating Enzymes UbcP3, Ubc15, and Rhp6 Affect Transcriptional Silencing of the Mating-Type Region

    PubMed Central

    Sig Nielsen, Inga; Nielsen, Olaf; Murray, Johanne M.; Thon, Geneviève

    2002-01-01

    Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6. PMID:12456009

  19. The fission yeast ubiquitin-conjugating enzymes UbcP3, Ubc15, and Rhp6 affect transcriptional silencing of the mating-type region.

    PubMed

    Nielsen, Inga Sig; Nielsen, Olaf; Murray, Johanne M; Thon, Geneviève

    2002-08-01

    Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.

  20. Gene-Silencing-Induced Changes in Carbohydrate Conformation in Relation to Bioenergy Value and Carbohydrate Subfractions in Modeled Plant (Medicago sativa) with Down-Regulation of HB12 and TT8 Transcription Factors.

    PubMed

    Li, Xinxin; Hannoufa, Abdelali; Zhang, Yonggen; Yu, Peiqiang

    2016-05-13

    Gene silencing with RNA interference (RNAi) technology may be capable of modifying internal structure at a molecular level. This structural modification could affect biofunctions in terms of biodegradation, biochemical metabolism, and bioactive compound availability. The objectives of this study were to (1) Detect gene silencing-induced changes in carbohydrate molecular structure in an alfalfa forage (Medicago sativa spp. sativa: alfalfa) with down-regulation of genes that encode transcription factors TT8 and HB12; (2) Determine gene silencing-induced changes in nutrient bioutilization and bioavailability in the alfalfa forage (Medicago sativa); and (3) Quantify the correlation between gene silencing-induced molecular structure changes and the nutrient bioutilization and bioavailability in animals of ruminants. The experimental treatments included: T1 = Non-transgenic and no-gene silenced alfalfa forage (code "NT"); T2 = HB12-RNAi forage with HB12 gene down regulation (code "HB12"); T3 = TT8-RNAi forage with TT8 gene down regulation (code "TT8"). The HB12 and TT8 gene silencing-induced molecular structure changes were determined by non-invasive and non-destructive advanced molecular spectroscopy in a middle infrared radiation region that focused on structural, non-structural and total carbohydrate compounds. The nutrient bioutilization and bioavailability of the modified forage were determined using NRC-2001 system in terms of total digestive nutrient (TDN), truly digestible fiber (tdNDF), non-fiber carbohydrate (tdNDF), fatty acid (tdFA), crude protein (tdCP) and bioenergy profiles (digestible energy, metabolizable energy, net energy) for ruminants. The carbohydrate subfractions were evaluated using the updated CNCPS 6.0 system. The results showed that gene silencing significantly affected tdNFC (42.3 (NT) vs. 38.7 (HB12) vs. 37.4% Dry Matter (TT8); p = 0.016) and tdCP (20.8 (NT) vs. 19.4 (HB12) vs. 22.3% DM (TT8); p = 0.009). The gene-silencing also affected

  1. Gene-Silencing-Induced Changes in Carbohydrate Conformation in Relation to Bioenergy Value and Carbohydrate Subfractions in Modeled Plant (Medicago sativa) with Down-Regulation of HB12 and TT8 Transcription Factors

    PubMed Central

    Li, Xinxin; Hannoufa, Abdelali; Zhang, Yonggen; Yu, Peiqiang

    2016-01-01

    Gene silencing with RNA interference (RNAi) technology may be capable of modifying internal structure at a molecular level. This structural modification could affect biofunctions in terms of biodegradation, biochemical metabolism, and bioactive compound availability. The objectives of this study were to (1) Detect gene silencing-induced changes in carbohydrate molecular structure in an alfalfa forage (Medicago sativa spp. sativa: alfalfa) with down-regulation of genes that encode transcription factors TT8 and HB12; (2) Determine gene silencing-induced changes in nutrient bioutilization and bioavailability in the alfalfa forage (Medicago sativa); and (3) Quantify the correlation between gene silencing-induced molecular structure changes and the nutrient bioutilization and bioavailability in animals of ruminants. The experimental treatments included: T1 = Non-transgenic and no-gene silenced alfalfa forage (code “NT”); T2 = HB12-RNAi forage with HB12 gene down regulation (code “HB12”); T3 = TT8-RNAi forage with TT8 gene down regulation (code “TT8”). The HB12 and TT8 gene silencing-induced molecular structure changes were determined by non-invasive and non-destructive advanced molecular spectroscopy in a middle infrared radiation region that focused on structural, non-structural and total carbohydrate compounds. The nutrient bioutilization and bioavailability of the modified forage were determined using NRC-2001 system in terms of total digestive nutrient (TDN), truly digestible fiber (tdNDF), non-fiber carbohydrate (tdNDF), fatty acid (tdFA), crude protein (tdCP) and bioenergy profiles (digestible energy, metabolizable energy, net energy) for ruminants. The carbohydrate subfractions were evaluated using the updated CNCPS 6.0 system. The results showed that gene silencing significantly affected tdNFC (42.3 (NT) vs. 38.7 (HB12) vs. 37.4% Dry Matter (TT8); p = 0.016) and tdCP (20.8 (NT) vs. 19.4 (HB12) vs. 22.3% DM (TT8); p = 0.009). The gene-silencing also

  2. High-Stearic and High-Oleic Cottonseed Oils Produced by Hairpin RNA-Mediated Post-Transcriptional Gene Silencing1

    PubMed Central

    Liu, Qing; Singh, Surinder P.; Green, Allan G.

    2002-01-01

    We have genetically modified the fatty acid composition of cottonseed oil using the recently developed technique of hairpin RNA-mediated gene silencing to down-regulate the seed expression of two key fatty acid desaturase genes, ghSAD-1-encoding stearoyl-acyl-carrier protein Δ9-desaturase and ghFAD2-1-encoding oleoyl-phosphatidylcholine ω6-desaturase. Hairpin RNA-encoding gene constructs (HP) targeted against either ghSAD-1 or ghFAD2-1 were transformed into cotton (Gossypium hirsutum cv Coker 315). The resulting down-regulation of the ghSAD-1 gene substantially increased stearic acid from the normal levels of 2% to 3% up to as high as 40%, and silencing of the ghFAD2-1 gene resulted in greatly elevated oleic acid content, up to 77% compared with about 15% in seeds of untransformed plants. In addition, palmitic acid was significantly lowered in both high-stearic and high-oleic lines. Similar fatty acid composition phenotypes were also achieved by transformation with conventional antisense constructs targeted against the same genes, but at much lower frequencies than were achieved with the HP constructs. By intercrossing the high-stearic and high-oleic genotypes, it was possible to simultaneously down-regulate both ghSAD-1 and ghFAD2-1 to the same degree as observed in the individually silenced parental lines, demonstrating for the first time, to our knowledge, that duplex RNA-induced posttranslational gene silencing in independent genes can be stacked without any diminution in the degree of silencing. The silencing of ghSAD-1 and/or ghFAD2-1 to various degrees enables the development of cottonseed oils having novel combinations of palmitic, stearic, oleic, and linoleic contents that can be used in margarines and deep frying without hydrogenation and also potentially in high-value confectionery applications. PMID:12177486

  3. Distinct transcriptional control in major immunogenetic subsets of chronic lymphocytic leukemia exhibiting subset-biased global DNA methylation profiles

    PubMed Central

    Kanduri, Meena; Marincevic, Millaray; Halldórsdóttir, Anna M.; Mansouri, Larry; Junevik, Katarina; Ntoufa, Stavroula; Kultima, Hanna Göransson; Isaksson, Anders; Juliusson, Gunnar; Andersson, Per-Ola; Ehrencrona, Hans; Stamatopoulos, Kostas; Rosenquist, Richard

    2012-01-01

    Chronic lymphocytic leukemia (CLL) can be divided into prognostic subgroups based on the IGHV gene mutational status, and is further characterized by multiple subsets of cases with quasi-identical or stereotyped B cell receptors that also share clinical and biological features. We recently reported differential DNA methylation profiles in IGHV-mutated and IGHV-unmutated CLL subgroups. For the first time, we here explore the global methylation profiles of stereotyped subsets with different prognosis, by applying high-resolution methylation arrays on CLL samples from three major stereotyped subsets: the poor-prognostic subsets #1 (n = 15) and #2 (n = 9) and the favorable-prognostic subset #4 (n = 15). Overall, the three subsets exhibited significantly different methylation profiles, which only partially overlapped with those observed in our previous study according to IGHV gene mutational status. Specifically, gene ontology analysis of the differentially methylated genes revealed a clear enrichment of genes involved in immune response, such as B cell activation (e.g., CD80, CD86 and IL10), with higher methylation levels in subset #1 than subsets #2 and #4. Accordingly, higher expression of the co-stimulatory molecules CD80 and CD86 was demonstrated in subset #4 vs. subset #1, pointing to a key role for these molecules in the crosstalk of CLL subset #4 cells with the microenvironment. In summary, investigation of three prototypic, stereotyped CLL subsets revealed distinct DNA methylation profiles for each subset, which suggests subset-biased patterns of transcriptional control and highlights a key role for epigenetics during leukemogenesis. PMID:23154584

  4. Distinct transcriptional control in major immunogenetic subsets of chronic lymphocytic leukemia exhibiting subset-biased global DNA methylation profiles.

    PubMed

    Kanduri, Meena; Marincevic, Millaray; Halldórsdóttir, Anna M; Mansouri, Larry; Junevik, Katarina; Ntoufa, Stavroula; Kultima, Hanna Göransson; Isaksson, Anders; Juliusson, Gunnar; Andersson, Per-Ola; Ehrencrona, Hans; Stamatopoulos, Kostas; Rosenquist, Richard

    2012-12-01

    Chronic lymphocytic leukemia (CLL) can be divided into prognostic subgroups based on the IGHV gene mutational status, and is further characterized by multiple subsets of cases with quasi-identical or stereotyped B cell receptors that also share clinical and biological features. We recently reported differential DNA methylation profiles in IGHV-mutated and IGHV-unmutated CLL subgroups. For the first time, we here explore the global methylation profiles of stereotyped subsets with different prognosis, by applying high-resolution methylation arrays on CLL samples from three major stereotyped subsets: the poor-prognostic subsets #1 (n = 15) and #2 (n = 9) and the favorable-prognostic subset #4 (n = 15). Overall, the three subsets exhibited significantly different methylation profiles, which only partially overlapped with those observed in our previous study according to IGHV gene mutational status. Specifically, gene ontology analysis of the differentially methylated genes revealed a clear enrichment of genes involved in immune response, such as B cell activation (e.g., CD80, CD86 and IL10), with higher methylation levels in subset #1 than subsets #2 and #4. Accordingly, higher expression of the co-stimulatory molecules CD80 and CD86 was demonstrated in subset #4 vs. subset #1, pointing to a key role for these molecules in the crosstalk of CLL subset #4 cells with the microenvironment. In summary, investigation of three prototypic, stereotyped CLL subsets revealed distinct DNA methylation profiles for each subset, which suggests subset-biased patterns of transcriptional control and highlights a key role for epigenetics during leukemogenesis.

  5. Advances in plant gene silencing methods.

    PubMed

    Pandey, Prachi; Senthil-Kumar, Muthappa; Mysore, Kirankumar S

    2015-01-01

    Understanding molecular mechanisms of transcriptional and posttranscriptional gene silencing pathways in plants over the past decades has led to development of tools and methods for silencing a target gene in various plant species. In this review chapter, both the recent understanding of molecular basis of gene silencing pathways and advances in various widely used gene silencing methods are compiled. We also discuss the salient features of the different methods like RNA interference (RNAi) and virus-induced gene silencing (VIGS) and highlight their advantages and disadvantages. Gene silencing technology is constantly progressing as reflected by rapidly emerging new methods. A succinct discussion on the recently developed methods like microRNA-mediated virus-induced gene silencing (MIR-VIGS) and microRNA-induced gene silencing (MIGS) is also provided. One major bottleneck in gene silencing approaches has been the associated off-target silencing. The other hurdle has been the lack of a universal approach that can be applied to all plants. For example, we face hurdles like incompatibility of VIGS vectors with the host and inability to use MIGS for plant species which are not easily transformable. However, the overwhelming research in this direction reflects the scope for overcoming the short comings of gene silencing technology.

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

    PubMed Central

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

    1999-01-01

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

  7. Silencing of the Hsf gene, the transcriptional regulator of A. gambiae male accessory glands, inhibits the formation of the mating plug in mated females and disrupts their monogamous behaviour.

    PubMed

    Dottorini, Tania; Persampieri, Tania; Palladino, Pietro; Spaccapelo, Roberta; Crisanti, Andrea

    2012-11-01

    Discovering the molecular factors that shape the mating behaviour and the fertility of the mosquito Anopheles gambiae, the principal vector of human malaria, is regarded as critical to better understand its reproductive success as well as for identifying new leads for malaria control measures. In A. gambiae mating induces complex behavioural and physiological changes in the females, including refractoriness to subsequent mating and induction of egg-laying. In other insects including Drosophila a group of proteins named Accessory gland proteins (Acps), produced by males and transferred with sperm to the female reproductive tract, have been implicated in this post-mating response. Although Acps represent a set of promising candidates for unravelling the mating physiology, their role in inducing behavioural changes in mated A. gambiae females remains largely unknown. In this work, we demonstrate that a down-regulation of a large fraction of Acp genes via silencing of the Acp regulating transcription factor Hsf, abolishes the formation of mating plug in mated females and fails to induce refractoriness of mated female to subsequent inseminations. A significant fraction of females mated to Hsf silenced males (66%) failed to receive the mating plug though seminal fluid had been transferred as documented by the presence of spermatozoa in the female sperm storage organ. Furthermore, nearly all females (95%) mated to HSF-silenced males were re-inseminated when exposed to males carrying EGPF marked sperm. Our findings provide evidence showing that Acp genes regulated by the transcription factor HSF play a key role in the function of the male accessory glands.

  8. Arabidopsis HIT4, a regulator involved in heat-triggered reorganization of chromatin and release of transcriptional gene silencing, relocates from chromocenters to the nucleolus in response to heat stress.

    PubMed

    Wang, Lian-Chin; Wu, Jia-Rong; Hsu, Yi-Ju; Wu, Shaw-Jye

    2015-01-01

    Arabidopsis HIT4 is known to mediate heat-induced decondensation of chromocenters and release from transcriptional gene silencing (TGS) with no change in the level of DNA methylation. It is unclear whether HIT4 and MOM1, a well-known DNA methylation-independent transcriptional silencer, have overlapping regulatory functions. A hit4-1/mom1 double mutant strain was generated. Its nuclear morphology and TGS state were compared with those of wild-type, hit4-1, and mom1 plants. Fluorescent protein tagging was employed to track the fates of HIT4, hit4-1 and MOM1 in vivo under heat stress. HIT4- and MOM1-mediated TGS were distinguishable. Both HIT4 and MOM1 were localized normally to chromocenters. Under heat stress, HIT4 relocated to the nucleolus, whereas MOM1 dispersed with the chromocenters. hit4-1 was able to relocate to the nucleolus under heat stress, but its relocation was insufficient to trigger the decompaction of chromocenters. The hypersensitivity to heat associated with the impaired reactivation of TGS in hit4-1 was not alleviated by mom1-induced release from TGS. HIT4 delineates a novel and MOM1-independent TGS regulation pathway. The involvement of a currently unidentified component that links HIT4 relocation and the large-scale reorganization of chromatin, and which is essential for heat tolerance in plants is hypothesized.

  9. 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

  10. Tapping RNA silencing pathways for plant biotechnology.

    PubMed

    Frizzi, Alessandra; Huang, Shihshieh

    2010-08-01

    Plants have evolved a variety of gene silencing pathways mediated by small RNAs. Mostly 21 or 24 nt in size, these small RNAs repress the expression of sequence homologous genes at the transcriptional, post-transcriptional and translational levels. These pathways, also referred as RNA silencing pathways, play important roles in regulating growth and development as well as in response to both biotic and abiotic stress. Although the molecular basis of these complicated and interconnected pathways has become clear only in recent years, RNA silencing effects were observed and utilized in transgenic plants early in the plant biotechnology era, more than two decades ago. Today, with a better understanding of the pathways, various genetic engineering approaches have been developed to apply RNA silencing more effectively and broadly. In addition to summarizing the current models of RNA silencing, this review discusses examples of its potential uses and related issues concerning its application in plant biotechnology.

  11. Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription.

    PubMed

    Martell, Danya J; Joshi, Chandra P; Gaballa, Ahmed; Santiago, Ace George; Chen, Tai-Yen; Jung, Won; Helmann, John D; Chen, Peng

    2015-11-03

    Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.

  12. Transcriptional repression by Rev-erbA alpha is dependent on the signature motif and helix 5 in the ligand binding domain: silencing does not involve an interaction with N-CoR.

    PubMed Central

    Downes, M; Burke, L J; Muscat, G E

    1996-01-01

    Rev-erbA alpha is an orphan nuclear receptor that functions as a dominant transcriptional repressor. Tissue culture and in situ hybridisation studies indicated that Rev-erbA alpha plays an important role in mammalian differentiation and development. Previous studies have localised the silencing domain of Rev-erbA alpha to the D/E region of the orphan receptor. This study utilised the GAL4 hybrid system to demonstrate that efficient repression is mediated by 34 amino acids (aa) between aa 455 and 488 in the E region of the receptor. This domain contains the ligand binding domain (LBD)-signature motif [(F/W)AKxxxxFxxLxxxDQxxLL] and a region that, according to the recently published crystal structures of steroid receptors, would be predicted to form helix 5 of the canonical LBD structure. Fine deletions and site-specific mutagenesis indicated that both the LBD signature motif and helix 5 were necessary for efficient silencing. Utilising mammalian two hybrid technology, we have also demonstrated that Rev-erbA alpha does not associate with the interaction domain (aa 2218-2451) of the nuclear receptor corepressor, N-CoR, that is known to interact with the thyroid hormone and retinoic acid receptors. This suggested that transcriptional repression by Rev-erbA alpha is not mediated through an interaction with N-CoR. In conclusion, we have identified and characterised the minimal domain of Rev-erbA alpha, that mediates transcriptional repression by this orphan receptor. PMID:8836173

  13. Silencing polygalacturonase expression inhibits tomato petiole abscission.

    PubMed

    Jiang, Cai-Zhong; Lu, Feng; Imsabai, Wachiraya; Meir, Shimon; Reid, Michael S

    2008-01-01

    Virus-induced gene silencing (VIGS) was used as a tool for functional analysis of cell wall-associated genes that have been suggested to be involved in leaf abscission. Tobacco rattle virus is an effective vector for VIGS in tomato (Lycopersicon esculentum). Silencing was more efficient when the plants were grown at 22 degrees C than when they were grown at 20 degrees C or 25 degrees C. The photobleaching phenotype resulting from silencing phytoene desaturase varied, depending on cultivar, from barely visible to photobleaching of entire leaves. To study the function of abscission-related genes, a purple transgenic tomato line constitutively expressing the maize anthocyanin regulatory gene, Leaf colour (Lc) was used. Silencing Lc expression in this line resulted in reduced anthocyanin production (reversing the colour from purple to green), thus providing a convenient silencing 'reporter'. Silencing tomato abscission-related polygalacturonases (TAPGs), using a TAPG1 fragment, delayed abscission and increased break strength of the abscission zone in explants treated with 1 mul l(-1) ethylene. The abundance of TAPG1 transcripts in the green (silenced) abscission zone tissues was <1% of that in the purple (non-silenced) controls. As a highly homologous region was used for all five polygalacturonases it is assumed that the effect of delayed abscission is the result of silencing all the genes in this family. By contrast, silencing abscission-related expansins (LeEXP11 and LeEXP12) and endoglucanases (LeCEL1 and LeCEL2) had no discernible effect on break strength, even when the two endoglucanase genes were silenced concurrently. Simultaneous silencing of TAPG and LeCEL1 was no more effective than silencing TAPG alone. The data demonstrate the importance of TAPGs in the abscission of leaf petioles.

  14. Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber, Apostichopus japonicus (Selenka), during aestivation

    NASA Astrophysics Data System (ADS)

    Wang, Tianming; Yang, Hongsheng; Zhao, Huan; Chen, Muyan; Wang, Bing

    2011-11-01

    The sea cucumber, Apostichopus japonicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling (DNA (cytosine-5)-methyltransferase 1, Methyl-CpG-binding domain protein 2), and Chromodomain-helicase-DNA-binding protein 5) was significantly higher during aestivation (Days 20 and 40). Similarly, we observed an increase in the expression of genes involved in histone acetylation (Histone deacetylase 3) and Histone-binding protein RBBP4) during the early (Days 5 and 10) and late phases (Days 20 and 40) of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers (Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5) was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.

  15. Practising Silence in Teaching

    ERIC Educational Resources Information Center

    Forrest, Michelle

    2013-01-01

    The concept "silence" has diametrically opposed meanings; it connotes peace and contemplation as well as death and oblivion. Silence can also be considered a practice. There is keeping the rule of silence to still the mind and find inner truth, as well as forcibly silencing in the sense of subjugating another to one's own purposes.…

  16. Practising Silence in Teaching

    ERIC Educational Resources Information Center

    Forrest, Michelle

    2013-01-01

    The concept "silence" has diametrically opposed meanings; it connotes peace and contemplation as well as death and oblivion. Silence can also be considered a practice. There is keeping the rule of silence to still the mind and find inner truth, as well as forcibly silencing in the sense of subjugating another to one's own purposes.…

  17. On the Mechanism of Gene Silencing in Saccharomyces cerevisiae

    PubMed Central

    Steakley, David Lee; Rine, Jasper

    2015-01-01

    Multiple mechanisms have been proposed for gene silencing in Saccharomyces cerevisiae, ranging from steric occlusion of DNA binding proteins from their recognition sequences in silenced chromatin to a specific block in the formation of the preinitiation complex to a block in transcriptional elongation. This study provided strong support for the steric occlusion mechanism by the discovery that RNA polymerase of bacteriophage T7 could be substantially blocked from transcribing from its cognate promoter when embedded in silenced chromatin. Moreover, unlike previous suggestions, we found no evidence for stalled RNA polymerase II within silenced chromatin. The effectiveness of the Sir protein–based silencing mechanism to block transcription activated by Gal4 at promoters in the domain of silenced chromatin was marginal, yet it improved when tested against mutant forms of the Gal4 protein, highlighting a role for specific activators in their sensitivity to gene silencing. PMID:26082137

  18. Small RNA-Mediated Epigenetic Myostatin Silencing.

    PubMed

    Roberts, Thomas C; Andaloussi, Samir El; Morris, Kevin V; McClorey, Graham; Wood, Matthew Ja

    2012-05-15

    Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

  19. Temporal Transcriptional Profiling of Somatic and Germ Cells Reveals Biased Lineage Priming of Sexual Fate in the Fetal Mouse Gonad

    PubMed Central

    Jameson, Samantha A.; Natarajan, Anirudh; Cool, Jonah; DeFalco, Tony; Maatouk, Danielle M.; Mork, Lindsey; Munger, Steven C.; Capel, Blanche

    2012-01-01

    The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates. PMID:22438826

  20. Transcript profiling of two alfalfa genotypes with contrasting cell wall composition in stems using a cross-species platform: optimizing analysis by masking biased probes

    PubMed Central

    2010-01-01

    Background The GeneChip® Medicago Genome Array, developed for Medicago truncatula, is a suitable platform for transcript profiling in tetraploid alfalfa [Medicago sativa (L.) subsp. sativa]. However, previous research involving cross-species hybridization (CSH) has shown that sequence variation between two species can bias transcript profiling by decreasing sensitivity (number of expressed genes detected) and the accuracy of measuring fold-differences in gene expression. Results Transcript profiling using the Medicago GeneChip® was conducted with elongating stem (ES) and post-elongation stem (PES) internodes from alfalfa genotypes 252 and 1283 that differ in stem cell wall concentrations of cellulose and lignin. A protocol was developed that masked probes targeting inter-species variable (ISV) regions of alfalfa transcripts. A probe signal intensity threshold was selected that optimized both sensitivity and accuracy. After masking for both ISV regions and previously identified single-feature polymorphisms (SFPs), the number of differentially expressed genes between the two genotypes in both ES and PES internodes was approximately 2-fold greater than the number detected prior to masking. Regulatory genes, including transcription factor and receptor kinase genes that may play a role in development of secondary xylem, were significantly over-represented among genes up-regulated in 252 PES internodes compared to 1283 PES internodes. Several cell wall-related genes were also up-regulated in genotype 252 PES internodes. Real-time quantitative RT-PCR of differentially expressed regulatory and cell wall-related genes demonstrated increased sensitivity and accuracy after masking for both ISV regions and SFPs. Over 1,000 genes that were differentially expressed in ES and PES internodes of genotypes 252 and 1283 were mapped onto putative orthologous loci on M. truncatula chromosomes. Clustering simulation analysis of the differentially expressed genes suggested co

  1. dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid.

    PubMed

    Lau, Su-Ee; Schwarzacher, Trude; Othman, Rofina Yasmin; Harikrishna, Jennifer Ann

    2015-08-11

    The R2R3-MYB genes regulate pigmentation and morphogenesis of flowers, including flower and cell shape, and therefore have importance in the development of new varieties of orchids. However, new variety development is limited by the long breeding time required in orchids. In this study, we identified a cDNA, DhMYB1, that is expressed during flower development in a hybrid orchid, Dendrobium hybrida (Dendrobium bobby messina X Dendrobium chao phraya) then used the direct application of dsRNA to observe the effect of gene silencing on flower phenotype and floral epidermal cell shape. Flower bud development in the Dendrobium hybrid was characterised into seven stages and the time of meiosis was determined as between stages 3 to 5 when the bud is approximately half of the mature size. Scanning electron microscopy characterisation of adaxial epidermal cells of the flower perianth, showed that the petals and sepals each are divided into two distinct domains based on cell shape and size, while the labellum comprises seven domains. Thirty-two partial cDNA fragments representing R2R3-MYB gene sequences were isolated from D. hybrida. Phylogenetic analysis revealed that nine of the translated sequences were clustered with MYB sequences that are known to be involved in cell shape development and from these, DhMYB1 was selected for full length cDNA cloning and functional study. Direct application of a 430 bp dsRNA from the 3' region of DhMYB1 to emerging orchid flower buds reduced expression of DhMYB1 RNA compared with untreated control. Scanning electron microscopy of adaxial epidermal cells within domain one of the labellum of flowers treated with DhMYB1 dsRNA showed flattened epidermal cells whilst those of control flowers were conical. DhMYB1 is expressed throughout flower bud development and is involved in the development of the conical cell shape of the epidermal cells of the Dendrobium hybrida flower labellum. The direct application of dsRNA changed the phenotype of

  2. New Construct Approaches for Efficient Gene Silencing in Plants

    PubMed Central

    Yan, Hua; Chretien, Robert; Ye, Jingsong; Rommens, Caius M.

    2006-01-01

    An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts. PMID:16766670

  3. "Schneebeli" and "Birger" silencers

    NASA Technical Reports Server (NTRS)

    Dollfus, Charles

    1923-01-01

    The Schneebeli silencer is made entirely of soft sheet steel. It is connected with the engine for receiving the exhaust gases, and consists of two parts: the silencer proper and a conical exit. The Birger silencer, made by the Ad Astra Company in Switzerland, is based on the principle of rapid cooling of the exhaust gases.

  4. Hitchcock's Melodramatic Silence.

    ERIC Educational Resources Information Center

    Hemmeter, Thomas

    1996-01-01

    Argues that the filmwork of Alfred Hitchcock shows his manipulation of melodramatic silence in that his films demonstrate a link between silence and truth. Concludes that in the simultaneous longing for and denial of the power of film silence lies the modernist complexity of Hitchcock's films that suggests the uses of melodramatic language in a…

  5. Posttranscriptional gene silencing in nuclei

    PubMed Central

    Hoffer, Paul; Ivashuta, Sergey; Pontes, Olga; Vitins, Alexa; Pikaard, Craig; Mroczka, Andrew; Wagner, Nicholas; Voelker, Toni

    2011-01-01

    In plants, small interfering RNAs (siRNAs) with sequence homology to transcribed regions of genes can guide the sequence-specific degradation of corresponding mRNAs, leading to posttranscriptional gene silencing (PTGS). The current consensus is that siRNA-mediated PTGS occurs primarily in the cytoplasm where target mRNAs are localized and translated into proteins. However, expression of an inverted-repeat double-stranded RNA corresponding to the soybean FAD2-1A desaturase intron is sufficient to silence FAD2-1, implicating nuclear precursor mRNA (pre-mRNA) rather than cytosolic mRNA as the target of PTGS. Silencing FAD2-1 using intronic or 3′-UTR sequences does not affect transcription rates of the target genes but results in the strong reduction of target transcript levels in the nucleus. Moreover, siRNAs corresponding to pre-mRNA–specific sequences accumulate in the nucleus. In Arabidopsis, we find that two enzymes involved in PTGS, Dicer-like 4 and RNA-dependent RNA polymerase 6, are localized in the nucleus. Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants. PMID:21173264

  6. Silencing of X-Linked MicroRNAs by Meiotic Sex Chromosome Inactivation

    PubMed Central

    Royo, Hélène; Seitz, Hervé; ElInati, Elias; Peters, Antoine H. F. M.; Stadler, Michael B.; Turner, James M. A.

    2015-01-01

    During the pachytene stage of meiosis in male mammals, the X and Y chromosomes are transcriptionally silenced by Meiotic Sex Chromosome Inactivation (MSCI). MSCI is conserved in therian mammals and is essential for normal male fertility. Transcriptomics approaches have demonstrated that in mice, most or all protein-coding genes on the X chromosome are subject to MSCI. However, it is unclear whether X-linked non-coding RNAs behave in a similar manner. The X chromosome is enriched in microRNA (miRNA) genes, with many exhibiting testis-biased expression. Importantly, high expression levels of X-linked miRNAs (X-miRNAs) have been reported in pachytene spermatocytes, indicating that these genes may escape MSCI, and perhaps play a role in the XY-silencing process. Here we use RNA FISH to examine X-miRNA expression in the male germ line. We find that, like protein-coding X-genes, X-miRNAs are expressed prior to prophase I and are thereafter silenced during pachynema. X-miRNA silencing does not occur in mouse models with defective MSCI. Furthermore, X-miRNAs are expressed at pachynema when present as autosomally integrated transgenes. Thus, we conclude that silencing of X-miRNAs during pachynema in wild type males is MSCI-dependent. Importantly, misexpression of X-miRNAs during pachynema causes spermatogenic defects. We propose that MSCI represents a chromosomal mechanism by which X-miRNAs, and other potential X-encoded repressors, can be silenced, thereby regulating genes with critical late spermatogenic functions. PMID:26509798

  7. Silencing of X-Linked MicroRNAs by Meiotic Sex Chromosome Inactivation.

    PubMed

    Royo, Hélène; Seitz, Hervé; ElInati, Elias; Peters, Antoine H F M; Stadler, Michael B; Turner, James M A

    2015-10-01

    During the pachytene stage of meiosis in male mammals, the X and Y chromosomes are transcriptionally silenced by Meiotic Sex Chromosome Inactivation (MSCI). MSCI is conserved in therian mammals and is essential for normal male fertility. Transcriptomics approaches have demonstrated that in mice, most or all protein-coding genes on the X chromosome are subject to MSCI. However, it is unclear whether X-linked non-coding RNAs behave in a similar manner. The X chromosome is enriched in microRNA (miRNA) genes, with many exhibiting testis-biased expression. Importantly, high expression levels of X-linked miRNAs (X-miRNAs) have been reported in pachytene spermatocytes, indicating that these genes may escape MSCI, and perhaps play a role in the XY-silencing process. Here we use RNA FISH to examine X-miRNA expression in the male germ line. We find that, like protein-coding X-genes, X-miRNAs are expressed prior to prophase I and are thereafter silenced during pachynema. X-miRNA silencing does not occur in mouse models with defective MSCI. Furthermore, X-miRNAs are expressed at pachynema when present as autosomally integrated transgenes. Thus, we conclude that silencing of X-miRNAs during pachynema in wild type males is MSCI-dependent. Importantly, misexpression of X-miRNAs during pachynema causes spermatogenic defects. We propose that MSCI represents a chromosomal mechanism by which X-miRNAs, and other potential X-encoded repressors, can be silenced, thereby regulating genes with critical late spermatogenic functions.

  8. Silencing transposable elements in the Drosophila germline.

    PubMed

    Yang, Fu; Xi, Rongwen

    2017-02-01

    Transposable elements or transposons are DNA pieces that can move around within the genome and are, therefore, potential threat to genome stability and faithful transmission of the genetic information in the germline. Accordingly, self-defense mechanisms have evolved in the metazoan germline to silence transposons, and the primary mechanism requires the germline-specific non-coding small RNAs, named Piwi-interacting RNA (piRNAs), which are in complex with Argonaute family of PIWI proteins (the piRNA-RISC complexes), to silence transposons. piRNA-mediated transposon silencing occurs at both transcriptional and post-transcriptional levels. With the advantages of genetic manipulation and advances of sequencing technology, much progress has been made on the molecular mechanisms of piRNA-mediated transposon silencing in Drosophila melanogaster, which will be the focus of this review. Because piRNA-mediated transposon silencing is evolutionarily conserved in metazoan, model organisms, such as Drosophila, will continue to be served as pioneer systems towards the complete understanding of transposon silencing in the metazoan germline.

  9. Transcriptional silencing of Cyclooxygenase-2 by hyper-methylation of the 5' CpG island in human gastric carcinoma cells.

    PubMed

    Song, S H; Jong, H S; Choi, H H; Inoue, H; Tanabe, T; Kim, N K; Bang, Y J

    2001-06-01

    It has been well established that overexpression of Cyclooxygenase-2 (Cox-2) in epithelial cells inhibits apoptosis and increases the invasiveness of malignant cells, favoring tumorigenesis and metastasis. However, the molecular mechanism that regulates Cox-2 expression has not been well defined in gastric carcinoma. In this study, we examined whether the Cox-2 expression could be regulated by hyper-methylation of the Cox-2 CpG island (spanning from -590 to +186 with respect to the transcription initiation site) in human gastric carcinoma cell lines. By Southern analysis, we found that three gastric cells (SNU-601, -620, and -719) without Cox-2 expression demonstrated hyper-methylation at the Cox-2 CpG island. A detailed methylation pattern using bisulfite sequencing analysis revealed that all of the CpG sites were completely methylated in SNU-601. Treatment with demethylating agents effectively reactivated the expression of Cox-2 and restored IL-1beta sensitivity in the previously resistant SNU-601. By transient transfection experiments, we demonstrate that constitutively active Cox-2 promoter activities were exhibited even without an exogenous stimulation in SNU-601. Furthermore, when the motif of the nuclear factor for interleukin-6 expression site, the cyclic AMP response element, or both was subjected to point mutation, the constitutive luciferase activity was markedly reduced. In addition, Cox-2 promoter activity was completely blocked by in vitro methylation of all of the CpG sites in the Cox-2 promoter region with SssI (CpG) methylase in SNU-601. Taken together, these results indicate that transcriptional repression of Cox-2 is caused by hyper-methylation of the Cox-2 CpG island in gastric carcinoma cell lines.

  10. Personalized gene silencing therapeutics for Huntington disease.

    PubMed

    Kay, C; Skotte, N H; Southwell, A L; Hayden, M R

    2014-07-01

    Gene silencing offers a novel therapeutic strategy for dominant genetic disorders. In specific diseases, selective silencing of only one copy of a gene may be advantageous over non-selective silencing of both copies. Huntington disease (HD) is an autosomal dominant disorder caused by an expanded CAG trinucleotide repeat in the Huntingtin gene (HTT). Silencing both expanded and normal copies of HTT may be therapeutically beneficial, but preservation of normal HTT expression is preferred. Allele-specific methods can selectively silence the mutant HTT transcript by targeting either the expanded CAG repeat or single nucleotide polymorphisms (SNPs) in linkage disequilibrium with the expansion. Both approaches require personalized treatment strategies based on patient genotypes. We compare the prospect of safe treatment of HD by CAG- and SNP-specific silencing approaches and review HD population genetics used to guide target identification in the patient population. Clinical implementation of allele-specific HTT silencing faces challenges common to personalized genetic medicine, requiring novel solutions from clinical scientists and regulatory authorities.

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

    PubMed

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

    2017-03-01

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

  12. [Correlation on a cellular level of gene transcriptional silencing and heterochromatin compartment dragging in case of PEV-producing eu-heterochromatin rearrangement in Drosophila melanogaster].

    PubMed

    Lavrov, S A; Shatskikh, A S; Kibanov, M V; Gvozdev, V A

    2013-01-01

    Eu-heterochromatic rearrangements transfer genes into the heterochromatin and cause their variegated inactivation (PEV). Genes affected by PEV often demonstrate association with heterochromatic nuclear compartment (a distinct area composed of heterochromatin sequences like satellite DNA and enriched in specific chromatin proteins e.g. HP1). Here, we investigate the nuclear localization and the expression levels of the genes subjected to PEV caused by chromosome inversion, In(2)A4. We demonstrate that the degree of PEV-caused gene inactivation depends on a developmental stage, and the maximum of repression corresponds to the gene expression activation period. In the case of In(2)A4 rearrangement we detect the dragging of affected euchromatic region into heterochromatic nuclear compartment and the increase in HP1 occupancy in this region. We developed a protocol of simultaneous RNA-DNA-protein staining to demonstrate firstly in a single cell a strong correlation between transcriptional activity of affected gene and its distance from chromosome 2 satellite DNA.

  13. Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor

    NASA Astrophysics Data System (ADS)

    Chen, Kuan-I.; Pan, Chien-Yuan; Li, Keng-Hui; Huang, Ying-Chih; Lu, Chia-Wei; Tang, Chuan-Yi; Su, Ya-Wen; Tseng, Ling-Wei; Tseng, Kun-Chang; Lin, Chi-Yun; Chen, Chii-Dong; Lin, Shih-Shun; Chen, Yit-Tsong

    2015-11-01

    Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3‧-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.

  14. Post-transcriptional silencing of CCR3 downregulates IL-4 stimulated release of eotaxin-3 (CCL26) and other CCR3 ligands in alveolar type II cells.

    PubMed

    Taka, Equar; Errahali, Younes J; Abonyo, Barack O; Bauer, David M; Heiman, Ann S

    2008-12-01

    Trafficking and inflammation in airway diseases are, in part, modulated by members of the CC chemokine family, eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26), which transduce signals through their CCR3 receptor. In this context, we hypothesized that transfecting alveolar type II epithelial cells with CCR3-targeted siRNA or antisense (AS-ODN) sequences will downregulate cellular synthesis and release of the primary CCR3 ligands CCL26 and CCL24 and will modulate other CCR3 ligands. The human A549 alveolar type II epithelium-like cell culture model was used for transfection and subsequent effects on CCR3 agonists. siRNAs were particularly effective. PCR showed a 60-80% decrease in mRNA and immunoblots showed up to 75-84% reduction of CCR3 in siRNA treated cells. CCR3-siRNA treatments reduced IL-4 stimulated CCL26 release and constitutive CCL24 release by 65% and 80%, respectively. Release of four additional CCR3 agonists RANTES, MCP-2, MCP-3 and MCP-4 was also significantly reduced by CCR3-siRNA treatments of the alveolar type II cells. Activation of eosinophils, assessed as superoxide anion generation, was reduced when eosinophils were treated with supernatants of A549 cells pretreated with CCR3-targeted siRNAs or AS-ODNs. Collectively, the data suggest that post-transcriptional regulation of CCR3 receptors may be a potential therapeutic approach for interrupting proinflammatory signaling.

  15. Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor.

    PubMed

    Chen, Kuan-I; Pan, Chien-Yuan; Li, Keng-Hui; Huang, Ying-Chih; Lu, Chia-Wei; Tang, Chuan-Yi; Su, Ya-Wen; Tseng, Ling-Wei; Tseng, Kun-Chang; Lin, Chi-Yun; Chen, Chii-Dong; Lin, Shih-Shun; Chen, Yit-Tsong

    2015-11-30

    Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3'-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.

  16. Optimal viral strategies for bypassing RNA silencing.

    PubMed

    Rodrigo, Guillermo; Carrera, Javier; Jaramillo, Alfonso; Elena, Santiago F

    2011-02-06

    The RNA silencing pathway constitutes a defence mechanism highly conserved in eukaryotes, especially in plants, where the underlying working principle relies on the repressive action triggered by the intracellular presence of double-stranded RNAs. This immune system performs a post-transcriptional suppression of aberrant mRNAs or viral RNAs by small interfering RNAs (siRNAs) that are directed towards their target in a sequence-specific manner. However, viruses have evolved strategies to escape from silencing surveillance while promoting their own replication. Several viruses encode suppressor proteins that interact with different elements of the RNA silencing pathway and block it. The different suppressors are not phylogenetically nor structurally related and also differ in their mechanism of action. Here, we adopt a model-driven forward-engineering approach to understand the evolution of suppressor proteins and, in particular, why viral suppressors preferentially target some components of the silencing pathway. We analysed three strategies characterized by different design principles: replication in the absence of a suppressor, suppressors targeting the first protein component of the pathway and suppressors targeting the siRNAs. Our results shed light on the question of whether a virus must opt for devoting more time into transcription or into translation and on which would be the optimal step of the silencing pathway to be targeted by suppressors. In addition, we discussed the evolutionary implications of such designing principles.

  17. RNAi induced gene silencing in crop improvement.

    PubMed

    Sinha, Subodh Kumar

    2010-12-01

    The RNA silencing is one of the innovative and efficient molecular biology tools to harness the down-regulation of expression of gene(s) specifically. To accomplish such selective modification of gene expression of a particular trait, homology dependent gene silencing uses a stunning variety of gene silencing viz. co-suppression, post-transcriptional gene silencing, virus-induced gene silencing etc. This family of diverse molecular phenomena has a common exciting feature of gene silencing which is collectively called RNA interference abbreviated to as RNAi. This molecular phenomenon has become a focal point of plant biology and medical research throughout the world. As a result, this technology has turned out to be a powerful tool in understanding the function of individual gene and has ultimately led to the tremendous use in crop improvement. This review article illustrates the application of RNAi in a broad area of crop improvement where this technology has been successfully used. It also provides historical perspective of RNAi discovery and its contemporary phenomena, mechanism of RNAi pathway.

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

    PubMed

    Gulec, Sukru; Collins, James F

    2014-01-01

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

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

    PubMed Central

    Gulec, Sukru; Collins, James F.

    2014-01-01

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

  20. The Epigenetic Pathways to Ribosomal DNA Silencing

    PubMed Central

    Srivastava, Rakesh; Srivastava, Rashmi

    2016-01-01

    SUMMARY Heterochromatin is the transcriptionally repressed portion of eukaryotic chromatin that maintains a condensed appearance throughout the cell cycle. At sites of ribosomal DNA (rDNA) heterochromatin, epigenetic states contribute to gene silencing and genome stability, which are required for proper chromosome segregation and a normal life span. Here, we focus on recent advances in the epigenetic regulation of rDNA silencing in Saccharomyces cerevisiae and in mammals, including regulation by several histone modifications and several protein components associated with the inner nuclear membrane within the nucleolus. Finally, we discuss the perturbations of rDNA epigenetic pathways in regulating cellular aging and in causing various types of diseases. PMID:27250769

  1. Abundance of female-biased and paucity of male-biased somatically expressed genes on the mouse X-chromosome.

    PubMed

    Reinius, Björn; Johansson, Martin M; Radomska, Katarzyna J; Morrow, Edward H; Pandey, Gaurav K; Kanduri, Chandrasekhar; Sandberg, Rickard; Williams, Robert W; Jazin, Elena

    2012-11-10

    Empirical evaluations of sexually dimorphic expression of genes on the mammalian X-chromosome are needed to understand the evolutionary forces and the gene-regulatory mechanisms controlling this chromosome. We performed a large-scale sex-bias expression analysis of genes on the X-chromosome in six different somatic tissues from mouse. Our results show that the mouse X-chromosome is enriched with female-biased genes and depleted of male-biased genes. This suggests that feminisation as well as de-masculinisation of the X-chromosome has occurred in terms of gene expression in non-reproductive tissues. Several mechanisms may be responsible for the control of female-biased expression on chromosome X, and escape from X-inactivation is a main candidate. We confirmed escape in case of Tmem29 using RNA-FISH analysis. In addition, we identified novel female-biased non-coding transcripts located in the same female-biased cluster as the well-known coding X-inactivation escapee Kdm5c, likely transcribed from the transition-region between active and silenced domains. We also found that previously known escapees only partially explained the overrepresentation of female-biased X-genes, particularly for tissue-specific female-biased genes. Therefore, the gene set we have identified contains tissue-specific escapees and/or genes controlled by other sexually skewed regulatory mechanisms. Analysis of gene age showed that evolutionarily old X-genes (>100 myr, preceding the radiation of placental mammals) are more frequently female-biased than younger genes. Altogether, our results have implications for understanding both gene regulation and gene evolution of mammalian X-chromosomes, and suggest that the final result in terms of the X-gene composition (masculinisation versus feminisation) is a compromise between different evolutionary forces acting on reproductive and somatic tissues.

  2. Abundance of female-biased and paucity of male-biased somatically expressed genes on the mouse X-chromosome

    PubMed Central

    2012-01-01

    Background Empirical evaluations of sexually dimorphic expression of genes on the mammalian X-chromosome are needed to understand the evolutionary forces and the gene-regulatory mechanisms controlling this chromosome. We performed a large-scale sex-bias expression analysis of genes on the X-chromosome in six different somatic tissues from mouse. Results Our results show that the mouse X-chromosome is enriched with female-biased genes and depleted of male-biased genes. This suggests that feminisation as well as de-masculinisation of the X-chromosome has occurred in terms of gene expression in non-reproductive tissues. Several mechanisms may be responsible for the control of female-biased expression on chromosome X, and escape from X-inactivation is a main candidate. We confirmed escape in case of Tmem29 using RNA-FISH analysis. In addition, we identified novel female-biased non-coding transcripts located in the same female-biased cluster as the well-known coding X-inactivation escapee Kdm5c, likely transcribed from the transition-region between active and silenced domains. We also found that previously known escapees only partially explained the overrepresentation of female-biased X-genes, particularly for tissue-specific female-biased genes. Therefore, the gene set we have identified contains tissue-specific escapees and/or genes controlled by other sexually skewed regulatory mechanisms. Analysis of gene age showed that evolutionarily old X-genes (>100 myr, preceding the radiation of placental mammals) are more frequently female-biased than younger genes. Conclusion Altogether, our results have implications for understanding both gene regulation and gene evolution of mammalian X-chromosomes, and suggest that the final result in terms of the X-gene composition (masculinisation versus feminisation) is a compromise between different evolutionary forces acting on reproductive and somatic tissues. PMID:23140559

  3. Nucleoporin Mediated Nuclear Positioning and Silencing of HMR

    PubMed Central

    Ruben, Giulia J.; Kirkland, Jacob G.; MacDonough, Tracy; Chen, Miao; Dubey, Rudra N.; Gartenberg, Marc R.; Kamakaka, Rohinton T.

    2011-01-01

    The organization of chromatin domains in the nucleus is an important factor in gene regulation. In eukaryotic nuclei, transcriptionally silenced chromatin clusters at the nuclear periphery while transcriptionally poised chromatin resides in the nuclear interior. Recent studies suggest that nuclear pore proteins (NUPs) recruit loci to nuclear pores to aid in insulation of genes from silencing and during gene activation. We investigated the role of NUPs at a native yeast insulator and show that while NUPs localize to the native tDNA insulator adjacent to the silenced HMR domain, loss of pore proteins does not compromise insulation. Surprisingly we find that NUPs contribute to silencing at HMR and are able to restore silencing to a silencing-defective HMR allele when tethered to the locus. We show that the perinuclear positioning of heterochromatin is important for the NUP-mediated silencing effect and find that loss of NUPs result in decreased localization of HMR to the nuclear periphery. We also show that loss of telomeric tethering pathways does not eliminate NUP localization to HMR, suggesting that NUPs may mediate an independent pathway for HMR association with the nuclear periphery. We propose that localization of NUPs to the tDNA insulator at HMR helps maintain the intranuclear position of the silent locus, which in turn contributes to the fidelity of silencing at HMR. PMID:21818277

  4. The neuron-restrictive silencer element: A dual enhancer/silencer crucial for patterned expression of a nicotinic receptor gene in the brain

    PubMed Central

    Bessis, Alain; Champtiaux, Nicolas; Chatelin, Laurent; Changeux, Jean-Pierre

    1997-01-01

    The neuron-restrictive silencer element (NRSE) has been identified in several neuronal genes and confers neuron specificity by silencing transcription in nonneuronal cells. NRSE is present in the promoter of the neuronal nicotinic acetylcholine receptor β2-subunit gene that determines its neuron-specific expression in the nervous system. Using transgenic mice, we show that NRSE may either silence or enhance transcription depending on the cellular context within the nervous system. In vitro in neuronal cells, NRSE activates transcription of synthetic promoters when located downstream in the 5′ untranslated region, or at less than 50 bp upstream from the TATA box, but switches to a silencer when located further upstream. In contrast, in nonneuronal cells NRSE always functions as a silencer. Antisense RNA inhibition shows that the NRSE-binding protein REST contributes to the activation of transcription in neuronal cells. PMID:9159173

  5. Rate of Amino Acid Substitution Is Influenced by the Degree and Conservation of Male-Biased Transcription Over 50 Myr of Drosophila Evolution

    PubMed Central

    Grath, Sonja; Parsch, John

    2012-01-01

    Sex-biased gene expression (i.e., the differential expression of genes between males and females) is common among sexually reproducing species. However, genes often differ in their sex-bias classification or degree of sex bias between species. There is also an unequal distribution of sex-biased genes (especially male-biased genes) between the X chromosome and the autosomes. We used whole-genome expression data and evolutionary rate estimates for two different Drosophilid lineages, melanogaster and obscura, spanning an evolutionary time scale of around 50 Myr to investigate the influence of sex-biased gene expression and chromosomal location on the rate of molecular evolution. In both lineages, the rate of protein evolution correlated positively with the male/female expression ratio. Genes with highly male-biased expression, genes expressed specifically in male reproductive tissues, and genes with conserved male-biased expression over long evolutionary time scales showed the fastest rates of evolution. An analysis of sex-biased gene evolution in both lineages revealed evidence for a “fast-X” effect in which the rate of evolution was greater for X-linked than for autosomal genes. This pattern was particularly pronounced for male-biased genes. Genes located on the obscura “neo-X” chromosome, which originated from a recent X-autosome fusion, showed rates of evolution that were intermediate between genes located on the ancestral X-chromosome and the autosomes. This suggests that the shift to X-linkage led to an increase in the rate of molecular evolution. PMID:22321769

  6. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  7. Multitasking of the piRNA Silencing Machinery: Targeting Transposable Elements and Foreign Genes in the Bdelloid Rotifer Adineta vaga

    PubMed Central

    Rodriguez, Fernando; Arkhipova, Irina R.

    2016-01-01

    RNA-mediated silencing processes play a key role in silencing of transposable elements, especially in the germ line, where piwi-interacting RNAs (piRNAs) are responsible for suppressing transposon mobility and maintaining genome integrity. We previously reported that the genome of Adineta vaga, the first sequenced representative of the phylum Rotifera (class Bdelloidea), is characterized by massive levels of horizontal gene transfer, by unusually low transposon content, and by highly diversified RNA-mediated silencing machinery. Here, we investigate genome-wide distribution of pi-like small RNAs, which in A. vaga are 25–31 nucleotides in length and have a strong 5′-uridine bias, while lacking ping-pong amplification signatures. In agreement with expectations, 71% of mapped reads corresponded to annotated transposons, with 93% of these reads being in the antisense orientation. Unexpectedly, a significant fraction of piRNAs originate from predicted coding regions corresponding to genes of putatively foreign origin. The distribution of piRNAs across foreign genes is not biased toward 3′-UTRs, instead resembling transposons in uniform distribution pattern throughout the gene body, and in predominantly antisense orientation. We also find that genes with small RNA coverage, including a number of genes of metazoan origin, are characterized by higher occurrence of telomeric repeats in the surrounding genomic regions, and by higher density of transposons in the vicinity, which have the potential to promote antisense transcription. Our findings highlight the complex interplay between RNA-based silencing processes and acquisition of genes at the genome periphery, which can result either in their loss or eventual domestication and integration into the host genome. PMID:27017627

  8. Multitasking of the piRNA Silencing Machinery: Targeting Transposable Elements and Foreign Genes in the Bdelloid Rotifer Adineta vaga.

    PubMed

    Rodriguez, Fernando; Arkhipova, Irina R

    2016-05-01

    RNA-mediated silencing processes play a key role in silencing of transposable elements, especially in the germ line, where piwi-interacting RNAs (piRNAs) are responsible for suppressing transposon mobility and maintaining genome integrity. We previously reported that the genome of Adineta vaga, the first sequenced representative of the phylum Rotifera (class Bdelloidea), is characterized by massive levels of horizontal gene transfer, by unusually low transposon content, and by highly diversified RNA-mediated silencing machinery. Here, we investigate genome-wide distribution of pi-like small RNAs, which in A. vaga are 25-31 nucleotides in length and have a strong 5'-uridine bias, while lacking ping-pong amplification signatures. In agreement with expectations, 71% of mapped reads corresponded to annotated transposons, with 93% of these reads being in the antisense orientation. Unexpectedly, a significant fraction of piRNAs originate from predicted coding regions corresponding to genes of putatively foreign origin. The distribution of piRNAs across foreign genes is not biased toward 3'-UTRs, instead resembling transposons in uniform distribution pattern throughout the gene body, and in predominantly antisense orientation. We also find that genes with small RNA coverage, including a number of genes of metazoan origin, are characterized by higher occurrence of telomeric repeats in the surrounding genomic regions, and by higher density of transposons in the vicinity, which have the potential to promote antisense transcription. Our findings highlight the complex interplay between RNA-based silencing processes and acquisition of genes at the genome periphery, which can result either in their loss or eventual domestication and integration into the host genome. Copyright © 2016 by the Genetics Society of America.

  9. Mariner Transposons Contain a Silencer: Possible Role of the Polycomb Repressive Complex 2

    PubMed Central

    Beauclair, Linda; Moiré, Nathalie; Arensbuger, Peter; Bigot, Yves

    2016-01-01

    Transposable elements are driving forces for establishing genetic innovations such as transcriptional regulatory networks in eukaryotic genomes. Here, we describe a silencer situated in the last 300 bp of the Mos1 transposase open reading frame (ORF) which functions in vertebrate and arthropod cells. Functional silencers are also found at similar locations within three other animal mariner elements, i.e. IS630-Tc1-mariner (ITm) DD34D elements, Himar1, Hsmar1 and Mcmar1. These silencers are able to impact eukaryotic promoters monitoring strong, moderate or low expression as well as those of mariner elements located upstream of the transposase ORF. We report that the silencing involves at least two transcription factors (TFs) that are conserved within animal species, NFAT-5 and Alx1. These cooperatively act with YY1 to trigger the silencing activity. Four other housekeeping transcription factors (TFs), neuron restrictive silencer factor (NRSF), GAGA factor (GAF) and GTGT factor (GTF), were also found to have binding sites within mariner silencers but their impact in modulating the silencer activity remains to be further specified. Interestingly, an NRSF binding site was found to overlap a 30 bp motif coding a highly conserved PHxxYSPDLAPxD peptide in mariner transposases. We also present experimental evidence that silencing is mainly achieved by co-opting the host Polycomb Repressive Complex 2 pathway. However, we observe that when PRC2 is impaired another host silencing pathway potentially takes over to maintain weak silencer activity. Mariner silencers harbour features of Polycomb Response Elements, which are probably a way for mariner elements to self-repress their transcription and mobility in somatic and germinal cells when the required TFs are expressed. At the evolutionary scale, mariner elements, through their exaptation, might have been a source of silencers playing a role in the chromatin configuration in eukaryotic genomes. PMID:26939020

  10. Silent Pedagogy and Rethinking Classroom Practice: Structuring Teaching through Silence Rather than Talk

    ERIC Educational Resources Information Center

    Ollin, Ros

    2008-01-01

    Classroom observations are an important source of information about teaching and about the practice of particular teachers. The paper considers the value placed on talk as opposed to silence in this context and suggests that a cultural bias towards talk means that silence is commonly perceived negatively. The paper is based on a qualitative…

  11. Silent Pedagogy and Rethinking Classroom Practice: Structuring Teaching through Silence Rather than Talk

    ERIC Educational Resources Information Center

    Ollin, Ros

    2008-01-01

    Classroom observations are an important source of information about teaching and about the practice of particular teachers. The paper considers the value placed on talk as opposed to silence in this context and suggests that a cultural bias towards talk means that silence is commonly perceived negatively. The paper is based on a qualitative…

  12. Sea urchin mtDBP is a two-faced transcription termination factor with a biased polarity depending on the RNA polymerase.

    PubMed

    Fernandez-Silva, P; Polosa, P L; Roberti, M; Di Ponzio, B; Gadaleta, M N; Montoya, J; Cantatore, P

    2001-11-15

    The sea urchin mitochondrial displacement (D)-loop binding protein mtDBP has been previously identified and cloned. The polypeptide (348 amino acids) displays a significant homology with the human mitochondrial transcription termination factor mTERF. This similarity, and the observation that the 3' ends of mitochondrial RNAs coded by opposite strands mapped in correspondence of mtDBP-binding sites, suggested that mtDBP could function as transcription termination factor in sea urchin mitochondria. To investigate such a role we tested the capability of mtDBP bound to its target sequence in the main non-coding region to affect RNA elongation by mitochondrial and bacteriophage T3 and T7 RNA polymerases. We show that mtDBP was able to terminate transcription bidirectionally when initiated by human mitochondrial RNA polymerase but only unidirectionally when initiated by T3 or T7 RNA polymerases. Time-course experiments indicated that mtDBP promotes true transcription termination rather than transcription pausing. These results indicate that mtDBP is able to function as a bipolar transcription termination factor in sea urchin mitochondria. The functional significance of such an activity could be linked to the previously proposed dual role of the protein in modulating mitochondrial DNA transcription and replication.

  13. RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence.

    PubMed

    Pumplin, Nathan; Voinnet, Olivier

    2013-11-01

    RNA silencing is a central regulator of gene expression in most eukaryotes and acts both at the transcriptional level through DNA methylation and at the post-transcriptional level through direct mRNA interference mediated by small RNAs. In plants and invertebrates, the same pathways also function directly in host defence against viruses by targeting viral RNA for degradation. Successful viruses have consequently evolved diverse mechanisms to avoid silencing, most notably through the expression of viral suppressors of RNA silencing. RNA silencing suppressors have also been recently identified in plant pathogenic bacteria and oomycetes, suggesting that disruption of host silencing is a general virulence strategy across several kingdoms of plant pathogens. There is also increasing evidence that plants have evolved specific defences against RNA-silencing suppression by pathogens, providing yet another illustration of the never-ending molecular arms race between plant pathogens and their hosts.

  14. On Observing Student Silence

    ERIC Educational Resources Information Center

    Amundrud, Thomas

    2011-01-01

    This article uses conversation analysis (CA) to look at how students in an advanced EGAP (English for general academic purposes) course discussion test create and manage the silence of a group member during the 7-min session. This is combined with a personal narrative inquiry, coinspired by autoethnography, on the author's participation in the…

  15. The Gift of Silence

    ERIC Educational Resources Information Center

    Haskins, Cathleen

    2011-01-01

    Slowing down, quieting the mind and body, and experiencing silence nourishes the spirit. Montessori educators are mandated to cultivate not just the intellect but the whole child. They recognize that nurturing the spirit of the child is part of what makes this form of education work so well. This article discusses the benefits of stillness and…

  16. The Gift of Silence

    ERIC Educational Resources Information Center

    Haskins, Cathleen

    2011-01-01

    Slowing down, quieting the mind and body, and experiencing silence nourishes the spirit. Montessori educators are mandated to cultivate not just the intellect but the whole child. They recognize that nurturing the spirit of the child is part of what makes this form of education work so well. This article discusses the benefits of stillness and…

  17. On Observing Student Silence

    ERIC Educational Resources Information Center

    Amundrud, Thomas

    2011-01-01

    This article uses conversation analysis (CA) to look at how students in an advanced EGAP (English for general academic purposes) course discussion test create and manage the silence of a group member during the 7-min session. This is combined with a personal narrative inquiry, coinspired by autoethnography, on the author's participation in the…

  18. Altered promoter nucleosome positioning is an early event in gene silencing.

    PubMed

    Hesson, Luke B; Sloane, Mathew A; Wong, Jason Wh; Nunez, Andrea C; Srivastava, Sameer; Ng, Benedict; Hawkins, Nicholas J; Bourke, Michael J; Ward, Robyn L

    2014-10-01

    Gene silencing in cancer frequently involves hypermethylation and dense nucleosome occupancy across promoter regions. How a promoter transitions to this silent state is unclear. Using colorectal adenomas, we investigated nucleosome positioning, DNA methylation, and gene expression in the early stages of gene silencing. Genome-wide gene expression correlated with highly positioned nucleosomes upstream and downstream of a nucleosome-depleted transcription start site (TSS). Hypermethylated promoters displayed increased nucleosome occupancy, specifically at the TSS. We investigated 2 genes, CDH1 and CDKN2B, which were silenced in adenomas but lacked promoter hypermethylation. Instead, silencing correlated with loss of nucleosomes from the -2 position upstream of the TSS relative to normal mucosa. In contrast, permanent CDH1 silencing in carcinoma cells was characterized by promoter hypermethylation and dense nucleosome occupancy. Our findings suggest that silenced genes transition through an intermediary stage involving altered promoter nucleosome positioning, before permanent silencing by hypermethylation and dense nucleosome occupancy.

  19. Silence: a double-edged sword.

    PubMed

    Bunkers, Sandra Schmidt

    2013-01-01

    The author in this column discusses the concept silence and the contradictions present with silence. Silence can be experienced in the following life patterns: silence as a pattern of surrendering to moments of awakening; silence as a pattern of bearing witness to life story; and, silence as a pattern of betrayal of trust. Each of these patterns is explored with their relevance for nursing.

  20. Successive silencing of tandem reporter genes in potato (Solanum tuberosum) over 5 years of vegetative propagation.

    PubMed

    Nocarova, Eva; Opatrny, Zdenek; Fischer, Lukas

    2010-10-01

    Transgenic plants represent an excellent tool for experimental plant biology and are an important component of modern agriculture. Fully understanding the stability of transgene expression is critical in this regard. Most changes in transgene expression occur soon after transformation and thus unwanted lines can be discarded easily; however, transgenes can be silenced long after their integration. To study the long-term changes in transgene expression in potato (Solanum tuberosum), the activity of two reporter genes, encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII), was monitored in a set of 17 transgenic lines over 5 years of vegetative propagation in vitro. A decrease in transgene expression was observed mainly in lines with higher initial GFP expression and a greater number of T-DNA insertions. Complete silencing of the reporter genes was observed in four lines (nearly 25 %), all of which successively silenced the two reporter genes, indicating an interconnection between their silencing. The loss of GFP fluorescence always preceded the loss of kanamycin resistance. Treatment with the demethylation drug 5-azacytidine indicated that silencing of the NPTII gene, but probably not of GFP, occurred directly at the transcriptional level. Successive silencing of the two reporter genes was also reproduced in lines with reactivated expression of previously silenced transgenes. We suggest a hypothetical mechanism involving the successive silencing of the two reporter genes that involves the switch of GFP silencing from the post-transcriptional to transcriptional level and subsequent spreading of methylation to the NPTII gene.

  1. Eliminating Bias

    EPA Pesticide Factsheets

    Learn how to eliminate bias from monitoring systems by instituting appropriate installation, operation, and quality assurance procedures. Provides links to download An Operator's Guide to Eliminating Bias in CEM Systems.

  2. Discovering host genes involved in the infection by the Tomato Yellow Leaf Curl Virus complex and in the establishment of resistance to the virus using Tobacco Rattle Virus-based post transcriptional gene silencing.

    PubMed

    Czosnek, Henryk; Eybishtz, Assaf; Sade, Dagan; Gorovits, Rena; Sobol, Iris; Bejarano, Eduardo; Rosas-Díaz, Tábata; Lozano-Durán, Rosa

    2013-03-22

    The development of high-throughput technologies allows for evaluating gene expression at the whole-genome level. Together with proteomic and metabolomic studies, these analyses have resulted in the identification of plant genes whose function or expression is altered as a consequence of pathogen attacks. Members of the Tomato yellow leaf curl virus (TYLCV) complex are among the most important pathogens impairing production of agricultural crops worldwide. To understand how these geminiviruses subjugate plant defenses, and to devise counter-measures, it is essential to identify the host genes affected by infection and to determine their role in susceptible and resistant plants. We have used a reverse genetics approach based on Tobacco rattle virus-induced gene silencing (TRV-VIGS) to uncover genes involved in viral infection of susceptible plants, and to identify genes underlying virus resistance. To identify host genes with a role in geminivirus infection, we have engineered a Nicotiana benthamiana line, coined 2IRGFP, which over-expresses GFP upon virus infection. With this system, we have achieved an accurate description of the dynamics of virus replication in space and time. Upon silencing selected N. benthamiana genes previously shown to be related to host response to geminivirus infection, we have identified eighteen genes involved in a wide array of cellular processes. Plant genes involved in geminivirus resistance were studied by comparing two tomato lines: one resistant (R), the other susceptible (S) to the virus. Sixty-nine genes preferentially expressed in R tomatoes were identified by screening cDNA libraries from infected and uninfected R and S genotypes. Out of the 25 genes studied so far, the silencing of five led to the total collapse of resistance, suggesting their involvement in the resistance gene network. This review of our results indicates that TRV-VIGS is an exquisite reverse genetics tool that may provide new insights into the molecular

  3. Discovering Host Genes Involved in the Infection by the Tomato Yellow Leaf Curl Virus Complex and in the Establishment of Resistance to the Virus Using Tobacco Rattle Virus-based Post Transcriptional Gene Silencing

    PubMed Central

    Czosnek, Henryk; Eybishtz, Assaf; Sade, Dagan; Gorovits, Rena; Sobol, Iris; Bejarano, Eduardo; Rosas-Díaz, Tábata; Lozano-Durán, Rosa

    2013-01-01

    The development of high-throughput technologies allows for evaluating gene expression at the whole-genome level. Together with proteomic and metabolomic studies, these analyses have resulted in the identification of plant genes whose function or expression is altered as a consequence of pathogen attacks. Members of the Tomato yellow leaf curl virus (TYLCV) complex are among the most important pathogens impairing production of agricultural crops worldwide. To understand how these geminiviruses subjugate plant defenses, and to devise counter-measures, it is essential to identify the host genes affected by infection and to determine their role in susceptible and resistant plants. We have used a reverse genetics approach based on Tobacco rattle virus-induced gene silencing (TRV-VIGS) to uncover genes involved in viral infection of susceptible plants, and to identify genes underlying virus resistance. To identify host genes with a role in geminivirus infection, we have engineered a Nicotiana benthamiana line, coined 2IRGFP, which over-expresses GFP upon virus infection. With this system, we have achieved an accurate description of the dynamics of virus replication in space and time. Upon silencing selected N. benthamiana genes previously shown to be related to host response to geminivirus infection, we have identified eighteen genes involved in a wide array of cellular processes. Plant genes involved in geminivirus resistance were studied by comparing two tomato lines: one resistant (R), the other susceptible (S) to the virus. Sixty-nine genes preferentially expressed in R tomatoes were identified by screening cDNA libraries from infected and uninfected R and S genotypes. Out of the 25 genes studied so far, the silencing of five led to the total collapse of resistance, suggesting their involvement in the resistance gene network. This review of our results indicates that TRV-VIGS is an exquisite reverse genetics tool that may provide new insights into the molecular

  4. Antiviral silencing in animals.

    PubMed

    Li, Hong-Wei; Ding, Shou-Wei

    2005-10-31

    RNA silencing or RNA interference (RNAi) refers to the small RNA-guided gene silencing mechanism conserved in a wide range of eukaryotic organisms from plants to mammals. As part of this special issue on the biology, mechanisms and applications of RNAi, here we review the recent advances on defining a role of RNAi in the responses of invertebrate and vertebrate animals to virus infection. Approximately 40 miRNAs and 10 RNAi suppressors encoded by diverse mammalian viruses have been identified. Assays used for the identification of viral suppressors and possible biological functions of both viral miRNAs and suppressors are discussed. We propose that herpes viral miRNAs may act as specificity factors to initiate heterochromatin assembly of the latent viral DNA genome in the nucleus.

  5. Transcript Profiling of Common Bean (Phaseolus vulgaris) Using the GeneChip Soybean Genome Array: Optimizing Analysis by Masking Biased Probes

    USDA-ARS?s Scientific Manuscript database

    Common bean (Phaseolus vulgaris) and soybean (Glycine max) both belong to the Phaseoleae tribe and share significant coding sequence homology. To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common b...

  6. The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 Gene Codes for an S-Adenosyl-l-Homocysteine Hydrolase Required for DNA Methylation-Dependent Gene Silencing

    PubMed Central

    Rocha, Pedro S.C.F.; Sheikh, Mazhar; Melchiorre, Rosalba; Fagard, Mathilde; Boutet, Stéphanie; Loach, Rebecca; Moffatt, Barbara; Wagner, Conrad; Vaucheret, Hervé; Furner, Ian

    2005-01-01

    Genes introduced into higher plant genomes can become silent (gene silencing) and/or cause silencing of homologous genes at unlinked sites (homology-dependent gene silencing or HDG silencing). Mutations of the HOMOLOGY-DEPENDENT GENE SILENCING1 (HOG1) locus relieve transcriptional gene silencing and methylation-dependent HDG silencing and result in genome-wide demethylation. The hog1 mutant plants also grow slowly and have low fertility and reduced seed germination. Three independent mutants of HOG1 were each found to have point mutations at the 3′ end of a gene coding for S-adenosyl-l-homocysteine (SAH) hydrolase, and hog1-1 plants show reduced SAH hydrolase activity. A transposon (hog1-4) and a T-DNA tag (hog1-5) in the HOG1 gene each behaved as zygotic embryo lethal mutants and could not be made homozygous. The results suggest that the homozygous hog1 point mutants are leaky and result in genome demethylation and poor growth and that homozygous insertion mutations result in zygotic lethality. Complementation of the hog1-1 point mutation with a T-DNA containing the gene coding for SAH hydrolase restored gene silencing, HDG silencing, DNA methylation, fast growth, and normal seed viability. The same T-DNA also complemented the zygotic embryo lethal phenotype of the hog1-4 tagged mutant. A model relating the HOG1 gene, DNA methylation, and methylation-dependent HDG silencing is presented. PMID:15659630

  7. Intergroup bias.

    PubMed

    Hewstone, Miles; Rubin, Mark; Willis, Hazel

    2002-01-01

    This chapter reviews the extensive literature on bias in favor of in-groups at the expense of out-groups. We focus on five issues and identify areas for future research: (a) measurement and conceptual issues (especially in-group favoritism vs. out-group derogation, and explicit vs. implicit measures of bias); (b) modern theories of bias highlighting motivational explanations (social identity, optimal distinctiveness, uncertainty reduction, social dominance, terror management); (c) key moderators of bias, especially those that exacerbate bias (identification, group size, status and power, threat, positive-negative asymmetry, personality and individual differences); (d) reduction of bias (individual vs. intergroup approaches, especially models of social categorization); and (e) the link between intergroup bias and more corrosive forms of social hostility.

  8. ATM Dependent Silencing Links Nucleolar Chromatin Reorganization to DNA Damage Recognition.

    PubMed

    Harding, Shane M; Boiarsky, Jonathan A; Greenberg, Roger A

    2015-10-13

    Resolution of DNA double-strand breaks (DSBs) is essential for the suppression of genome instability. DSB repair in transcriptionally active genomic regions represents a unique challenge that is associated with ataxia telangiectasia mutated (ATM) kinase-mediated transcriptional silencing. Despite emerging insights into the underlying mechanisms, how DSB silencing connects to DNA repair remains undefined. We observe that silencing within the rDNA depends on persistent DSBs. Non-homologous end-joining was the predominant mode of DSB repair allowing transcription to resume. ATM-dependent rDNA silencing in the presence of persistent DSBs led to the large-scale reorganization of nucleolar architecture, with movement of damaged chromatin to nucleolar cap regions. These findings identify ATM-dependent temporal and spatial control of DNA repair and provide insights into how communication between DSB signaling and ongoing transcription promotes genome integrity.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-04-09

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

  11. Highly efficient gene silencing using perfect complementary artificial miRNA targeting AP1 or heteromeric artificial miRNA targeting AP1 and CAL genes

    USDA-ARS?s Scientific Manuscript database

    Gene silencing is a useful technique for elucidating biological function of genes by knocking down their expression. Recently developed artificial microRNAs (amiRNAs) exploit an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs that target transcript...

  12. Rethinking the Day of Silence

    ERIC Educational Resources Information Center

    Murphy, Adriana

    2013-01-01

    Back in 2006, 7th and 8th graders at Green Acres, the K-8 independent school where the author taught in suburban Maryland, participated in the Day of Silence. The Day of Silence is a national event: Students across the country take a one-day pledge of silence to show that they want to make schools safe for all students, regardless of their sexual…

  13. Rethinking the Day of Silence

    ERIC Educational Resources Information Center

    Murphy, Adriana

    2013-01-01

    Back in 2006, 7th and 8th graders at Green Acres, the K-8 independent school where the author taught in suburban Maryland, participated in the Day of Silence. The Day of Silence is a national event: Students across the country take a one-day pledge of silence to show that they want to make schools safe for all students, regardless of their sexual…

  14. "Listening Silence" and Its Discursive Effects

    ERIC Educational Resources Information Center

    Applebaum, Barbara

    2016-01-01

    While researchers have studied how white silence protects white innocence and white ignorance, in this essay Barbara Applebaum explores a form of white silence that she refers to as "listening silence" in which silence protects white innocence but does not necessarily promote resistance to learning. White listening silence can appear to…

  15. "Listening Silence" and Its Discursive Effects

    ERIC Educational Resources Information Center

    Applebaum, Barbara

    2016-01-01

    While researchers have studied how white silence protects white innocence and white ignorance, in this essay Barbara Applebaum explores a form of white silence that she refers to as "listening silence" in which silence protects white innocence but does not necessarily promote resistance to learning. White listening silence can appear to…

  16. Yeast heterochromatin is a dynamic structure that requires silencers continuously

    PubMed Central

    Cheng, Tzu-Hao; Gartenberg, Marc R.

    2000-01-01

    Transcriptional silencing of the HM loci in yeast requires cis-acting elements, termed silencers, that function during S-phase passage to establish the silent state. To study the role of the regulatory elements in maintenance of repression, site-specific recombination was used to uncouple preassembled silent chromatin fragments from silencers. DNA rings excised from HMR were initially silent but ultimately reactivated, even in G1- or G2/M-arrested cells. In contrast, DNA rings bearing HML-derived sequence were stably repressed due to the presence of a protosilencing element. These data show that silencers (or protosilencers) are required continuously for maintenance of silent chromatin. Reactivation of unstably repressed rings was blocked by overexpression of silencing proteins Sir3p and Sir4p, and chromatin immunoprecipitation studies showed that overexpressed Sir3p was incorporated into silent chromatin. Importantly, the protein was incorporated even when expressed outside of S phase, during G1 arrest. That silencing factors can associate with and stabilize preassembled silent chromatin in non-S-phase cells demonstrates that heterochromatin in yeast is dynamic. PMID:10691737

  17. Antiviral RNA silencing suppression activity of Tomato spotted wilt virus NSs protein.

    PubMed

    Ocampo Ocampo, T; Gabriel Peralta, S M; Bacheller, N; Uiterwaal, S; Knapp, A; Hennen, A; Ochoa-Martinez, D L; Garcia-Ruiz, H

    2016-06-17

    In addition to regulating gene expression, RNA silencing is an essential antiviral defense system in plants. Triggered by double-stranded RNA, silencing results in degradation or translational repression of target transcripts. Viruses are inducers and targets of RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressors that interfere with this process, such as the Tomato spotted wilt virus (TSWV) NSs protein. The mechanism by which NSs suppresses RNA silencing and its role in viral infection and movement remain to be determined. We cloned NSs from the Hawaii isolate of TSWV and using two independent assays show for the first time that this protein restored pathogenicity and supported the formation of local infection foci by suppressor-deficient Turnip mosaic virus and Turnip crinkle virus. Demonstrating the suppression of RNA silencing directed against heterologous viruses establishes the foundation to determine the means used by NSs to block this antiviral process.

  18. Recent patents in RNA silencing in plants: constructs, methods and applications in plant biotechnology.

    PubMed

    López-Gomollón, Sara; Dalmay, Tamas

    2010-11-01

    RNA silencing is a recently discovered mechanism to regulate gene expression at transcriptional and posttranscriptional levels. It is based on the recognition and methylation of target genes or cleavage of target mRNAs by small RNA molecules, with length varying from 21 to 24 nucleotides. RNA silencing plays an important role modulating most of the important cell processes, such as growth, development or stress response. During the past few years, diverse strategies have been applied to exploit RNA silencing as a tool to create plants with enhanced economical properties or able to cope with pathogens or abiotic stress. This review describes the most important patents related to RNA silencing in plants, which disclose vectors designed to induce RNA silencing by hairpin RNAs, amplicons or virus-based plasmids, methods for detection and quantification of silencing as well as general uses in plant biotechnology.

  19. Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results

    PubMed Central

    Garcia-Bardon, Andreas

    2016-01-01

    Real-time reverse transcription polymerase chain reaction (PCR) is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed strong variations in cDNA yield. Absolute expression data in naïve and trauma settings varied substantially between these kits. Normalization with a housekeeping gene failed to reduce kit-dependent variations, whereas normalization eliminated differences when naïve samples from the same region were used. The study shows strong evidence that choice of commercial cDNA synthesis kit has a major impact on PCR results and, consequently, on comparability between studies. Additionally, it provides a solution to overcome this limitation by normalization with data from naïve samples. This simple step helps to compare mRNA expression data between different studies and groups. PMID:27898720

  20. Edwin Hubble's Silence

    NASA Astrophysics Data System (ADS)

    Lago, D.

    2013-04-01

    In late 1928 Edwin Hubble was right in the middle of using V. M. Slipher's redshift data to prove that the universe is expanding, when Hubble's boss, George Hale, directed him to drop everything and rush to the Grand Canyon and test it as a possible site for Hale's planned 200-inch telescope. On his way, Hubble stopped at Lowell Observatory and met with V. M. Slipher. The letters both men wrote about this visit suggest that Hubble never said a word about his being in the middle of using Slipher's research to transform the universe. At the least, this silence is symbolic of the silence with which astronomical history has often treated Slipher's work. A survey of the historical literature suggests several reasons for this. Theorists and observers in astronomy (and other sciences) have long had different perspectives about how science works, and those who place more importance on theory have tended to credit the idea of the expanding universe to the theorists. Also, many sources indicate that Edwin Hubble was not a modest man or generous about sharing credit.

  1. The 25–26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes

    PubMed Central

    Jia, Jinbu; Lu, Wenqin; Zhong, Chengcheng; Zhou, Ran; Xu, Junjie; Liu, Wei; Gou, Xiuhong; Wang, Qinhu; Yin, Junliang; Xu, Cheng; Shan, Weixing

    2017-01-01

    Small RNAs (sRNAs) are important non-coding RNA regulators, playing key roles in developmental regulation, transposon suppression, environmental response, host–pathogen interaction and other diverse biological processes. However, their roles in oomycetes are poorly understood. Here, we performed sRNA sequencing and RNA sequencing of Phytophthora parasitica at stages of vegetative growth and infection of Arabidopsis roots to examine diversity and function of sRNAs in P. parasitica, a model hemibiotrophic oomycete plant pathogen. Our results indicate that there are two distinct types of sRNA-generating loci in P. parasitica genome, giving rise to clusters of 25–26 nt and 21 nt sRNAs, respectively, with no significant strand-biases. The 25–26 nt sRNA loci lie predominantly in gene-sparse and repeat-rich regions, and overlap with over 7000 endogenous gene loci. These overlapped genes are typically P. parasitica species-specific, with no homologies to the sister species P. infestans. They include approximately 40% RXLR effector genes, 50% CRN effector genes and some elicitor genes. The transcripts of most of these genes could not be detected at both the vegetative mycelium and infection stages as revealed by RNA sequencing, indicating that the 25–26 nt sRNAs are associated with efficient silencing of these genes. The 21 nt sRNA loci typically overlap with the exon regions of highly expressed genes, suggesting that the biogenesis of the 21 nt sRNAs may be dependent on the level of gene transcription and that these sRNAs do not mediate efficient silencing of homologous genes. Analyses of the published P. infestans sRNA and mRNA sequencing data consistently show that the 25–26 nt sRNAs, but not the 21 nt sRNAs, may mediate efficient gene silencing in Phytophthora. PMID:28512457

  2. The 25-26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes.

    PubMed

    Jia, Jinbu; Lu, Wenqin; Zhong, Chengcheng; Zhou, Ran; Xu, Junjie; Liu, Wei; Gou, Xiuhong; Wang, Qinhu; Yin, Junliang; Xu, Cheng; Shan, Weixing

    2017-01-01

    Small RNAs (sRNAs) are important non-coding RNA regulators, playing key roles in developmental regulation, transposon suppression, environmental response, host-pathogen interaction and other diverse biological processes. However, their roles in oomycetes are poorly understood. Here, we performed sRNA sequencing and RNA sequencing of Phytophthora parasitica at stages of vegetative growth and infection of Arabidopsis roots to examine diversity and function of sRNAs in P. parasitica, a model hemibiotrophic oomycete plant pathogen. Our results indicate that there are two distinct types of sRNA-generating loci in P. parasitica genome, giving rise to clusters of 25-26 nt and 21 nt sRNAs, respectively, with no significant strand-biases. The 25-26 nt sRNA loci lie predominantly in gene-sparse and repeat-rich regions, and overlap with over 7000 endogenous gene loci. These overlapped genes are typically P. parasitica species-specific, with no homologies to the sister species P. infestans. They include approximately 40% RXLR effector genes, 50% CRN effector genes and some elicitor genes. The transcripts of most of these genes could not be detected at both the vegetative mycelium and infection stages as revealed by RNA sequencing, indicating that the 25-26 nt sRNAs are associated with efficient silencing of these genes. The 21 nt sRNA loci typically overlap with the exon regions of highly expressed genes, suggesting that the biogenesis of the 21 nt sRNAs may be dependent on the level of gene transcription and that these sRNAs do not mediate efficient silencing of homologous genes. Analyses of the published P. infestans sRNA and mRNA sequencing data consistently show that the 25-26 nt sRNAs, but not the 21 nt sRNAs, may mediate efficient gene silencing in Phytophthora.

  3. Transcript profiling of common bean (Phaseolus vulgaris L.) using the GeneChip® Soybean Genome Array: optimizing analysis by masking biased probes

    PubMed Central

    2010-01-01

    Background Common bean (Phaseolus vulgaris L.) and soybean (Glycine max) both belong to the Phaseoleae tribe and share significant coding sequence homology. This suggests that the GeneChip® Soybean Genome Array (soybean GeneChip) may be used for gene expression studies using common bean. Results To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate to the soybean GeneChip. Initial data analysis showed a decreased sensitivity and accuracy of measuring differential gene expression in common bean cross-species hybridization (CSH) GeneChip data compared to that of soybean. We employed a method that masked putative probes targeting inter-species variable (ISV) regions between common bean and soybean. A masking signal intensity threshold was selected that optimized both sensitivity and accuracy of measuring differential gene expression. After masking for ISV regions, the number of differentially-expressed genes identified in common bean was increased by 2.8-fold reflecting increased sensitivity. Quantitative RT-PCR (qRT-PCR) analysis of 20 randomly selected genes and purine-ureide pathway genes demonstrated an increased accuracy of measuring differential gene expression after masking for ISV regions. We also evaluated masked probe frequency per probe set to gain insight into the sequence divergence pattern between common bean and soybean. The sequence divergence pattern analysis suggested that the genes for basic cellular functions and metabolism were highly conserved between soybean and common bean. Additionally, our results show that some classes of genes, particularly those associated with environmental adaptation, are highly divergent. Conclusions The soybean GeneChip is a suitable cross-species platform for transcript profiling in common bean when used in combination with the masking protocol described. In addition to transcript

  4. GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells.

    PubMed

    Tchasovnikarova, Iva A; Timms, Richard T; Matheson, Nicholas J; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A; Lehner, Paul J

    2015-06-26

    Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.

  5. The emerging world of small silencing RNAs in protozoan parasites

    PubMed Central

    Atayde, Vanessa D.; Tschudi, Christian; Ullu, Elisabetta

    2011-01-01

    A new RNA world has emerged in the past 10 years with the discovery of a plethora of 20- to 30-nucleotide long small RNAs that are involved in various gene silencing mechanisms. These small RNAs have considerably changed our view of the regulation of gene expression in eukaryotic organisms, with a major shift towards epigenetic and post-transcriptional mechanisms. Here we focus on the striking diversity of small silencing RNAs that have been identified in a number of protozoan parasites and their potential biological role. PMID:21497553

  6. Silence, an Eye of Knowledge

    ERIC Educational Resources Information Center

    Aghamohammadi, Mehdi

    2017-01-01

    One of the conspicuous features of the twentieth-century West was silence. This idea could be supported by examining reflections of Ludwig Wittgenstein, Fritz Mauthner, John Cage, Samuel Beckett, Ihab Hassan, Franz Kafka, Wassily Kandinsky, Jean-Paul Sartre, Virginia Woolf, Wolfgang Iser, Jacques Derrida, and Pierre Macherey. To me, silence is not…

  7. Organizational Silence in Sports Employees

    ERIC Educational Resources Information Center

    Bastug, Gulsum; Pala, Adem; Yilmaz, Taner; Duyan, Mehdi; Gunel, Ilker

    2016-01-01

    Organizational silence can be defined as a way of behaviour belonging to men and women employees in the organization exhibited without reflecting their feelings, ideas, concerns and suggestions related with their workplaces, works for which they are responsible or other activities of the organization. In the period of organizational silence,…

  8. Silence Amenity Engineering

    NASA Astrophysics Data System (ADS)

    Fujita, Hajime

    Engineering civilization brought convenient and comfortable life to us. However, some environmental problems such as various pollutions have also been developed with it. Acoustical noise is one of the major problems in modern life. Noise is generated from a noise source and propagates through transmitting medium such as the air and eventually reaches a receiver, usually a human being. The noise problem can be avoided, therefore, if one of those three elements in the noise problem is removed completely. In actual case, engineers are looking for most efficient way combining the controls for these three elements. In this article, basic characteristics of noise is reviewed briefly at first, then sound field analysis to predict sound transmission is discussed Aerodynamic noise is one of the major problems in silence amenity engineering today. Basic concept of the aerodynamic noise generation mechanism is discussed in detail with applications to turbo-machinery and high speed train noise control technology.

  9. The eerie silence

    NASA Astrophysics Data System (ADS)

    Davies, Paul

    2010-03-01

    Whether or not we are alone in the universe is one of the great outstanding questions of existence. For thousands of years it was restricted to the realm of philosophy and theology, but 50 years ago it became part of science. In April 1960 a young US astronomer, Frank Drake, began using a radio telescope to investigate whether signals from an extraterrestrial community might be coming our way. Known as the Search for Extraterrestrial Intelligence, or SETI, it has grown into a major international enterprise, involving scientific institutions in several countries. Apart from a few oddities, however, all that the radio astronomers have encountered is an eerie silence. So is humankind the only technological civilization in the universe after all? Or might we be looking for the wrong thing in the wrong place at the wrong time?

  10. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    NASA Astrophysics Data System (ADS)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  11. In vivo chromatin accessibility correlates with gene silencing in Drosophila.

    PubMed Central

    Boivin, A; Dura, J M

    1998-01-01

    Gene silencing by heterochromatin is a well-known phenomenon that, in Drosophila, is called position effect variegation (PEV). The long-held hypothesis that this gene silencing is associated with an altered chromatin structure received direct support only recently. Another gene-silencing phenomenon in Drosophila, although similar in its phenotype of variegation, has been shown to be associated with euchromatic sequences and is dependent on developmental regulators of the Polycomb group (Pc-G) of gene products. One model proposes that the Pc-G products may cause a local heterochromatinization that maintains a repressed state of transcription of their target genes. Here, we test these models by measuring the accessibility of white or miniwhite sequences, in different contexts, to the Escherichia coli dam DNA methyltransferase in vivo. We present evidence that PEV and Pc-G-mediated repression mechanisms, although based on different protein factors, may indeed involve similar higher-order chromatin structure. PMID:9832530

  12. Histone deacetylase inhibitors reverse gene silencing in Friedreich's ataxia.

    PubMed

    Herman, David; Jenssen, Kai; Burnett, Ryan; Soragni, Elisabetta; Perlman, Susan L; Gottesfeld, Joel M

    2006-10-01

    Expansion of GAA x TTC triplets within an intron in FXN (the gene encoding frataxin) leads to transcription silencing, forming the molecular basis for the neurodegenerative disease Friedreich's ataxia. Gene silencing at expanded FXN alleles is accompanied by hypoacetylation of histones H3 and H4 and trimethylation of histone H3 at Lys9, observations that are consistent with a heterochromatin-mediated repression mechanism. We describe the synthesis and characterization of a class of histone deacetylase (HDAC) inhibitors that reverse FXN silencing in primary lymphocytes from individuals with Friedreich's ataxia. We show that these molecules directly affect the histones associated with FXN, increasing acetylation at particular lysine residues on histones H3 and H4 (H3K14, H4K5 and H4K12). This class of HDAC inhibitors may yield therapeutics for Friedreich's ataxia.

  13. Gene Silencing in Crustaceans: From Basic Research to Biotechnologies

    PubMed Central

    Sagi, Amir; Manor, Rivka; Ventura, Tomer

    2013-01-01

    Gene silencing through RNA interference (RNAi) is gaining momentum for crustaceans, both in basic research and for commercial development. RNAi has proven instrumental in a growing number of crustacean species, revealing the functionality of novel crustacean genes essential among others to development, growth, metabolism and reproduction. Extensive studies have also been done on silencing of viral transcripts in crustaceans, contributing to the understanding of the defense mechanisms of crustaceans and strategies employed by viruses to overcome these. The first practical use of gene silencing in aquaculture industry has been recently achieved, through manipulation of a crustacean insulin-like androgenic gland hormone. This review summarizes the advancements in the use of RNAi in crustaceans, and assesses the advantages of this method, as well as the current hurdles that hinder its large-scale practice. PMID:24705266

  14. MORC Family ATPases Required for Heterochromatin Condensation and Gene Silencing#

    PubMed Central

    Moissiard, Guillaume; Cokus, Shawn J.; Cary, Joshua; Feng, Suhua; Billi, Allison C.; Stroud, Hume; Husmann, Dylan; Zhan, Ye; Lajoie, Bryan R.; McCord, Rachel Patton; Hale, Christopher J.; Feng, Wei; Michaels, Scott D.; Frand, Alison R.; Pellegrini, Matteo; Dekker, Job; Kim, John K.; Jacobsen, Steve

    2012-01-01

    Transposable elements (TEs) and DNA repeats are commonly targeted by DNA and histone methylation to achieve epigenetic gene silencing. We isolated mutations in two Arabidopsis genes, AtMORC1 and AtMORC6, which cause de-repression of DNA-methylated genes and TEs, but no losses of DNA or histone methylation. AtMORC1 and AtMORC6 are members of the conserved Microrchidia (MORC) adenosine triphosphatase (ATPase) family, predicted to catalyze alterations in chromosome superstructure. The atmorc1 and atmorc6 mutants show decondensation of pericentromeric heterochromatin, increased interaction of pericentromeric regions with the rest of the genome, and transcriptional defects that are largely restricted to loci residing in pericentromeric regions. Knockdown of the single MORC homolog in Caenorhabditis elegans also impairs transgene silencing. We propose that the MORC ATPases are conserved regulators of gene silencing in eukaryotes. PMID:22555433

  15. Detailed Structural-Functional Analysis of the Krüppel-like Factor 16 (KLF16) Transcription Factor Reveals Novel Mechanisms for Silencing Sp/KLF Sites Involved in Metabolism and Endocrinology*

    PubMed Central

    Daftary, Gaurang S.; Lomberk, Gwen A.; Buttar, Navtej S.; Allen, Thomas W.; Grzenda, Adrienne; Zhang, Jinsan; Zheng, Ye; Mathison, Angela J.; Gada, Ravi P.; Calvo, Ezequiel; Iovanna, Juan L.; Billadeau, Daniel D.; Prendergast, Franklyn G.; Urrutia, Raul

    2012-01-01

    Krüppel-like factor (KLF) proteins have elicited significant attention due to their emerging key role in metabolic and endocrine diseases. Here, we extend this knowledge through the biochemical characterization of KLF16, unveiling novel mechanisms regulating expression of genes involved in reproductive endocrinology. We found that KLF16 selectively binds three distinct KLF-binding sites (GC, CA, and BTE boxes). KLF16 also regulated the expression of several genes essential for metabolic and endocrine processes in sex steroid-sensitive uterine cells. Mechanistically, we determined that KLF16 possesses an activation domain that couples to histone acetyltransferase-mediated pathways, as well as a repression domain that interacts with the histone deacetylase chromatin-remodeling system via all three Sin3 isoforms, suggesting a higher level of plasticity in chromatin cofactor selection. Molecular modeling combined with molecular dynamic simulations of the Sin3a-KLF16 complex revealed important insights into how this interaction occurs at an atomic resolution level, predicting that phosphorylation of Tyr-10 may modulate KLF16 function. Phosphorylation of KLF16 was confirmed by in vivo 32P incorporation and controlled by a Y10F site-directed mutant. Inhibition of Src-type tyrosine kinase signaling as well as the nonphosphorylatable Y10F mutation disrupted KLF16-mediated gene silencing, demonstrating that its function is regulatable rather than constitutive. Subcellular localization studies revealed that signal-induced nuclear translocation and euchromatic compartmentalization constitute an additional mechanism for regulating KLF16 function. Thus, this study lends insights on key biochemical mechanisms for regulating KLF sites involved in reproductive biology. These data also contribute to the new functional information that is applicable to understanding KLF16 and other highly related KLF proteins. PMID:22203677

  16. 5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level.

    PubMed

    Tyč, Dimitrij; Nocarová, Eva; Sikorová, Lenka; Fischer, Lukáš

    2017-08-01

    Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.

  17. Silencing a prohibitin alters plant development and senescence.

    PubMed

    Chen, Jen-Chih; Jiang, Cai-Zhong; Reid, Michael S

    2005-10-01

    Prohibitins, highly conserved mitochondrial proteins, have been shown to play important roles in cell cycling and senescence in animals and yeast. Sequences with high similarity to prohibitins have been identified in a number of plant species, but their function has not yet been demonstrated. The deduced amino acid sequences of PhPHB1 and PhPHB2, sequences that we identified in a petunia floral expressed sequence tag (EST) database, show high similarity to those of prohibitin-1 and prohibitin-2 proteins, respectively, reported from yeast, animals and plants. Southern analysis suggested that these genes were members of small gene families with at least two prohibitin-1 homologs and four prohibitin-2 homologs. When we downregulated expression of prohibitin-1 using a Tobacco rattle virus-based (TRV), virus-induced gene silencing system (VIGS), we observed plants with smaller and distorted leaves and flowers. Cells in silenced flowers were larger than in control flowers, indicating a substantial reduction in the number of cell divisions that took place during corolla development. The life of silenced flowers was shorter than that of controls, whether on the plant or detached. The respiration of silenced flowers was higher than that of controls, and we observed a marked increase in the abundance of transcripts of a catalase and a small heat-shock protein in the silenced flowers. Our data indicate that prohibitins play a key role in plant development and senescence.

  18. Silencing near tRNA genes is nucleosome-mediated and distinct from boundary element function

    PubMed Central

    Good, Paul D.; Kendall, Ann; Ignatz-Hoover, James; Miller, Erin L.; Pai, Dave A.; Rivera, Sara R.; Carrick, Brian; Engelke, David R.

    2013-01-01

    Transfer RNA (tRNA) genes and other RNA polymerase III transcription units are dispersed in high copy throughout nuclear genomes, and can antagonize RNA polymerase II transcription in their immediate chromosomal locus. Previous work in Saccharomyces cerevisiae found that this local silencing required subnuclear clustering of the tRNA genes near the nucleolus. Here we show that the silencing also requires nucleosome participation, though the nature of the nucleosome interaction appears distinct from other forms of transcriptional silencing. Analysis of an extensive library of histone amino acid substitutions finds a large number of residues that affect the silencing, both in the histone N-terminal tails and on the nucleosome disk surface. The residues on the disk surfaces involved are largely distinct from those affecting other regulatory phenomena. Consistent with the large number of histone residues affecting tgm silencing, survey of chromatin modification mutations shows that several enzymes known to affect nucleosome modification and positioning are also required. The enzymes include an Rpd3 deacetylase complex, Hos1 deacetylase, Glc7 phosphatase, and the RSC nucleosome remodeling activity, but not multiple other activities required for other silencing forms or boundary element function at tRNA gene loci. Models for communication between the tRNA gene transcription complexes and local chromatin are discussed. PMID:23707796

  19. Silencing near tRNA genes is nucleosome-mediated and distinct from boundary element function.

    PubMed

    Good, Paul D; Kendall, Ann; Ignatz-Hoover, James; Miller, Erin L; Pai, Dave A; Rivera, Sara R; Carrick, Brian; Engelke, David R

    2013-08-15

    Transfer RNA (tRNA) genes and other RNA polymerase III transcription units are dispersed in high copy throughout nuclear genomes, and can antagonize RNA polymerase II transcription in their immediate chromosomal locus. Previous work in Saccharomyces cerevisiae found that this local silencing required subnuclear clustering of the tRNA genes near the nucleolus. Here we show that the silencing also requires nucleosome participation, though the nature of the nucleosome interaction appears distinct from other forms of transcriptional silencing. Analysis of an extensive library of histone amino acid substitutions finds a large number of residues that affect the silencing, both in the histone N-terminal tails and on the nucleosome disk surface. The residues on the disk surfaces involved are largely distinct from those affecting other regulatory phenomena. Consistent with the large number of histone residues affecting tgm silencing, survey of chromatin modification mutations shows that several enzymes known to affect nucleosome modification and positioning are also required. The enzymes include an Rpd3 deacetylase complex, Hos1 deacetylase, Glc7 phosphatase, and the RSC nucleosome remodeling activity, but not multiple other activities required for other silencing forms or boundary element function at tRNA gene loci. Models for communication between the tRNA gene transcription complexes and local chromatin are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Silence and the Notion of the Commons.

    ERIC Educational Resources Information Center

    Franklin, Ursula

    1994-01-01

    Stresses the value of silence, the right to have silence, and how technology has manipulated the sound environment and therefore taken silence out of common availability. Discusses noise pollution and the manipulative use of sound for private gain. Suggests taking action to restore the right to silence. (LP)

  1. Silence and the Notion of the Commons.

    ERIC Educational Resources Information Center

    Franklin, Ursula

    1994-01-01

    Stresses the value of silence, the right to have silence, and how technology has manipulated the sound environment and therefore taken silence out of common availability. Discusses noise pollution and the manipulative use of sound for private gain. Suggests taking action to restore the right to silence. (LP)

  2. Phenotypic diversification by gene silencing in Phytophthora plant pathogens.

    PubMed

    Vetukuri, Ramesh R; Asman, Anna Km; Jahan, Sultana N; Avrova, Anna O; Whisson, Stephen C; Dixelius, Christina

    2013-11-01

    Advances in genome sequencing technologies have enabled generation of unprecedented information on genome content and organization. Eukaryote genomes in particular may contain large populations of transposable elements (TEs) and other repeated sequences. Active TEs can result in insertional mutations, altered transcription levels and ectopic recombination of DNA. The genome of the oomycete plant pathogen, Phytophthora infestans, contains vast numbers of TE sequences. There are also hundreds of predicted disease-promoting effector proteins, predominantly located in TE-rich genomic regions. Expansion of effector gene families is also a genomic signature of related oomycetes such as P. sojae. Deep sequencing of small RNAs (sRNAs) from P. infestans has identified sRNAs derived from all families of transposons, highlighting the importance of RNA silencing for maintaining these genomic invaders in an inactive form. Small RNAs were also identified from specific effector encoding genes, possibly leading to RNA silencing of these genes and variation in pathogenicity and virulence toward plant resistance genes. Similar findings have also recently been made for the distantly related species, P. sojae. Small RNA "hotspots" originating from arrays of amplified gene sequences, or from genes displaying overlapping antisense transcription, were also identified in P. infestans. These findings suggest a major role for RNA silencing processes in the adaptability and diversification of these economically important plant pathogens. Here we review the latest progress and understanding of gene silencing in oomycetes with emphasis on transposable elements and sRNA-associated events.

  3. Genome Reactivation after the Silence in Mitosis: Recapitulating Mechanisms of Development?

    PubMed Central

    Zaret, Kenneth S.

    2014-01-01

    Transcription is silenced during mitosis and re-activated at mitotic exit. The dynamics and identities of “bookmarking” transcription factors and chromatin marks that mediate reactivation often recapitulate that observed during cell identity establishment in development. Thus, features of post-mitotic gene re-activation can provide insights into mechanisms of developmental cell fate establishment. PMID:24780732

  4. How to use intentional silence.

    PubMed

    Kemerer, Douglas

    2016-09-07

    Rationale and key points This article explains intentional silence, which can provide a therapeutic nursing presence that demonstrates compassion and respect for the patient. » Intentional silence can enhance the therapeutic relationship between the nurse and patient. » Intentional silence can be used to reduce the patient's emotional lability by ensuring that they feel listened to. » It is important that nurses are able to respond therapeutically to rhetorical statements and/or those concerning serious or severe clinical circumstances. » The nurse can use intentional silence to support the patient in acknowledging, processing and reflecting on changes in their health. Reflective activity 'How to' articles can help to update your practice and ensure it remains evidence based. Apply this article to your practice. Reflect on and write a short account of: 1. How this article will change your practice. 2. How you could use this article to educate your colleagues. Subscribers can upload their reflective accounts at rcni.com/portfolio.

  5. Herpes Simplex Virus Type 1 Suppresses RNA-Induced Gene Silencing in Mammalian Cells▿

    PubMed Central

    Wu, Zetang; Zhu, Yali; Bisaro, David M.; Parris, Deborah S.

    2009-01-01

    RNA-induced silencing is a potent innate antiviral defense strategy in plants, and suppression of silencing is a hallmark of pathogenic plant viruses. However, the impact of silencing as a mammalian antiviral defense mechanism and the ability of mammalian viruses to suppress silencing in natural host cells have remained controversial. The ability of herpes simplex virus type 1 (HSV-1) to suppress silencing was examined in a transient expression system that employed an imperfect hairpin to target degradation of transcripts encoding enhanced green fluorescent protein (EGFP). HSV-1 infection suppressed EGFP-specific silencing as demonstrated by increased EGFP mRNA levels and an increase in the EGFP mRNA half-life. The increase in EGFP mRNA stability occurred despite the well-characterized host macromolecular shutoff functions of HSV-1 that globally destabilize mRNAs. Moreover, mutant viruses defective in these functions increased the stability of EGFP mRNA even more than did the wild-type virus in silenced cells compared to results in control cells. The importance of RNA silencing to HSV-1 replication was confirmed by a significantly enhanced virus burst size in cells in which silencing was knocked down with small inhibitory RNAs directed to Argonaute 2, an integral component of the silencing complex. Given that HSV-1 encodes several microRNAs, it is possible that a dynamic equilibrium exists between silencing and silencing suppression that is capable of modulating viral gene expression to promote replication, to evade host defenses, and/or to promote latency. PMID:19369325

  6. Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

    PubMed

    Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community.

  7. Artificial trans-acting siRNAs confer consistent and effective gene silencing.

    PubMed

    de la Luz Gutiérrez-Nava, Maria; Aukerman, Milo J; Sakai, Hajime; Tingey, Scott V; Williams, Robert W

    2008-06-01

    Manipulating gene expression is critical to exploring gene function and a useful tool for altering commercial traits. Techniques such as hairpin-based RNA interference, virus-induced gene silencing, and artificial microRNAs take advantage of endogenous posttranscriptional gene silencing pathways to block translation of designated transcripts. Here we present a novel gene silencing method utilizing artificial trans-acting small interfering RNAs in Arabidopsis (Arabidopsis thaliana). Replacing the endogenous small interfering RNAs encoded in the TAS1c gene with sequences from the FAD2 gene silenced FAD2 activity to levels comparable to the fad2-1 null allele in nearly all transgenic events. Interestingly, exchanging the endogenous miR173 target sequence in TAS1c with an miR167 target sequence led to variable, inefficient silencing of FAD2, suggesting a specific requirement for the miR173 trigger for production of small interfering RNAs from the TAS1c locus.

  8. Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

    PubMed

    Amabile, Angelo; Migliara, Alessandro; Capasso, Paola; Biffi, Mauro; Cittaro, Davide; Naldini, Luigi; Lombardo, Angelo

    2016-09-22

    Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.

  9. Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing.

    PubMed

    Chen, Chun-Kan; Blanco, Mario; Jackson, Constanza; Aznauryan, Erik; Ollikainen, Noah; Surka, Christine; Chow, Amy; Cerase, Andrea; McDonel, Patrick; Guttman, Mitchell

    2016-10-28

    The Xist long noncoding RNA orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the three-dimensional (3D) structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here, we show that Xist directly interacts with the Lamin B receptor, an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing by recruiting the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the 3D structure of DNA, enabling Xist and its silencing proteins to spread across the X to silence transcription. Copyright © 2016, American Association for the Advancement of Science.

  10. Polycomb-Mediated Gene Silencing in Arabidopsis thaliana

    PubMed Central

    Kim, Dong-Hwan; Sung, Sibum

    2014-01-01

    Polycomb group (PcG) proteins are conserved chromatin regulators involved in the control of key developmental programs in eukaryotes. They collectively provide the transcriptional memory unique to each cell identity by maintaining transcriptional states of developmental genes. PcG proteins form multi-protein complexes, known as Polycomb repressive complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2). PRC1 and PRC2 contribute to the stable gene silencing in part through catalyzing covalent histone modifications. Components of PRC1 and PRC2 are well conserved from plants to animals. PcG-mediated gene silencing has been extensively investigated in efforts to understand molecular mechanisms underlying developmental programs in eukaryotes. Here, we describe our current knowledge on PcG-mediated gene repression which dictates developmental programs by dynamic layers of regulatory activities, with an emphasis given to the model plant Arabidopsis thaliana. PMID:25410906

  11. Silencing of toxic gene expression by Fis

    PubMed Central

    Karambelkar, Shweta; Swapna, Ganduri; Nagaraja, Valakunja

    2012-01-01

    Bacteria and bacteriophages have evolved DNA modification as a strategy to protect their genomes. Mom protein of bacteriophage Mu modifies the phage DNA, rendering it refractile to numerous restriction enzymes and in turn enabling the phage to successfully invade a variety of hosts. A strong fortification, a combined activity of the phage and host factors, prevents untimely expression of mom and associated toxic effects. Here, we identify the bacterial chromatin architectural protein Fis as an additional player in this crowded regulatory cascade. Both in vivo and in vitro studies described here indicate that Fis acts as a transcriptional repressor of mom promoter. Further, our data shows that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. We propose that a combined repressive effect of Fis and previously characterized negative regulatory factors could be responsible to keep the gene silenced most of the time. We thus present a new facet of Fis function in Mu biology. In addition to bringing about overall downregulation of Mu genome, it also ensures silencing of the advantageous but potentially lethal mom gene. PMID:22287621

  12. Assessment of RNAi-induced silencing in banana (Musa spp.).

    PubMed

    Dang, Tuong Vi T; Windelinckx, Saskia; Henry, Isabelle M; De Coninck, Barbara; Cammue, Bruno P A; Swennen, Rony; Remy, Serge

    2014-09-18

    In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana. Using embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6-8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target

  13. Journal bias or author bias?

    PubMed

    Harris, Ian

    2016-01-01

    I read with interest the comment by Mark Wilson in the Indian Journal of Medical Ethics regarding bias and conflicts of interest in medical journals. Wilson targets one journal (the New England Journal of Medicine: NEJM) and one particular "scandal" to make his point that journals' decisions on publication are biased by commercial conflicts of interest (CoIs). It is interesting that he chooses the NEJM which, by his own admission, had one of the strictest CoI policies and had published widely on this topic. The feeling is that if the NEJM can be guilty, they can all be guilty.

  14. Characterization of Arabidopsis Genes Involved in Gene Silencing. Final Progress Report

    SciTech Connect

    Grant, S. R.

    1999-02-05

    Enhancer of gene silencing 1 (egs1) is an Arabidopsis mutant that enhances post-transcriptional gene silencing of the rolB gene introduced by genetic engineering (transgene). The goal of our proposal was cloning EGS1 based on its map position. Although we screened more than 2000 chromosomes for recombination, we were unable to get closer than 2 cM to the gene. We experienced an unexpected tendency of the post-transcriptionally silenced transgene to switch to a more stable silenced state. This made it impossible to select egs1 homozygotes for map based cloning. This forced us to reconsider our cloning strategy. One possibility would have been to use a different transgene as the target of gene silencing. We tested two other transgenes. Both encoded proteins unrelated to the first but they were all expressed from the same type of promoter and they all had a similar tendency to become post-transcriptionally silenced. After screening over 80 F2 segregants from each cross between our egs1 mutant and Arabidopsis of the same ecotype homozygous for the new transgene, we were disappointed to find that the egs1 mutation did not enhance post-transcription silencing of the two new genes. In 80 plants we expected to have between 4 and 6 plants that were homozygous for the transgene and for the mutant egs1 allele. If egs1 mutations could enhance gene silencing of the new transgene, these plants would not express it. However all the double homozygotes still expressed the transgene. Therefore, we could not change the target transgene for mapping. This was the state of the cloning at the time for renewal of the grant in 1999. Because the selection of new meaningful recombinant plants had become extremely inefficient using the original rolB transgene, we abandoned the attempt at map based cloning and did not apply for further funding.

  15. Design, Cloning, and In Vitro Screening of Artificial miRNAs to Silence Alpha-1 Antitrypsin.

    PubMed

    Borel, Florie; Mueller, Christian

    2017-01-01

    This protocol describes the design, cloning, and in vitro screening of artificial microRNAs (miRNAs) to silence alpha-1 antitrypsin (AAT). This method would be of interest to silence AAT in a variety of in vitro or in vivo models, and prevalidated sequences against human AAT are provided. This simple 5-day protocol may more generally be used to design artificial miRNAs against any transcript.

  16. The capacity of target silencing by Drosophila PIWI and piRNAs

    PubMed Central

    Post, Christina; Clark, Josef P.; Sytnikova, Yuliya A.; Chirn, Gung-Wei

    2014-01-01

    Although Piwi proteins and Piwi-interacting RNAs (piRNAs) genetically repress transposable elements (TEs), it is unclear how the highly diverse piRNA populations direct Piwi proteins to silence TE targets without silencing the entire transcriptome. To determine the capacity of piRNA-mediated silencing, we introduced reporter genes into Drosophila OSS cells, which express microRNAs (miRNAs) and piRNAs, and compared the Piwi pathway to the Argonaute pathway in gene regulation. Reporter constructs containing several target sites that were robustly silenced by miRNAs were not silenced to the same degrees by piRNAs. However, another set of reporters we designed to enable a large number of both TE-directed and genic piRNAs to bind were robustly silenced by the PIWI/piRNA complex in OSS cells. These reporters show that a bulk of piRNAs are required to pair to the reporter's transcripts and not the reporter's DNA sequence to engage PIWI-mediated silencing. Following our genome-wide study of PIWI-regulated targets in OSS cells, we assessed candidate gene elements with our reporter platform. These results suggest TE sequences are the most direct of PIWI regulatory targets while coding genes are less directly affected by PIWI targeting. Finally, our study suggests that the PIWI transcriptional silencing mechanism triggers robust chromatin changes on targets with sufficient piRNA binding, and preferentially regulates TE transcripts because protein-coding transcripts lack a threshold of targeting by piRNA populations. This reporter platform will facilitate future dissections of the PIWI-targeting mechanism. PMID:25336588

  17. Gene silencing triggered by non-LTR retrotransposons in the female germline of Drosophila melanogaster.

    PubMed Central

    Robin, Stéphanie; Chambeyron, Séverine; Bucheton, Alain; Busseau, Isabelle

    2003-01-01

    Several studies have recently shown that the activity of some eukaryotic transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent gene-silencing mechanisms in their regulation. Here we provide data indicating that two non-LTR retrotransposons of Drosophila melanogaster are themselves natural triggers of homology-dependent gene silencing. We show that, in the female germline of D. melanogaster, fragments from the R1 or from the I retrotransposons can mediate silencing of chimeric transcription units into which they are inserted. This silencing is probably mediated by sequence identity with endogenous copies of the retrotransposons because it does not occur with a fragment from the divergent R1 elements of Bombyx mori, and, when a fragment of I is used, it occurs only in females containing functional copies of the I element. This silencing is not accompanied by cosuppression of the endogenous gene homologous to the chimeric transcription unit, which contrasts to some other silencing mechanisms in Drosophila. These observations suggest that in the female germline of D. melanogaster the R1 and I retrotransposons may self-regulate their own activity and their copy number by triggering homology-dependent gene silencing. PMID:12807773

  18. C. elegans RNA-dependent RNA polymerases rrf-1 and ego-1 silence Drosophila transgenes by differing mechanisms.

    PubMed

    Duan, Guowen; Saint, Robert B; Helliwell, Chris A; Behm, Carolyn A; Wang, Ming-Bo; Waterhouse, Peter M; Gordon, Karl H J

    2013-04-01

    Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis.

  19. Organizational Silence and Hidden Threats to Patient Safety

    PubMed Central

    Henriksen, Kerm; Dayton, Elizabeth

    2006-01-01

    Organizational silence refers to a collective-level phenomenon of saying or doing very little in response to significant problems that face an organization. The paper focuses on some of the less obvious factors contributing to organizational silence that can serve as threats to patient safety. Converging areas of research from the cognitive, social, and organizational sciences and the study of sociotechnical systems help to identify some of the underlying factors that serve to shape and sustain organizational silence. These factors have been organized under three levels of analysis: (1) individual factors, including the availability heuristic, self-serving bias, and the status quo trap; (2) social factors, including conformity, diffusion of responsibility, and microclimates of distrust; and (3) organizational factors, including unchallenged beliefs, the good provider fallacy, and neglect of the interdependencies. Finally, a new role for health care leaders and managers is envisioned. It is one that places high value on understanding system complexity and does not take comfort in organizational silence. PMID:16898978

  20. Robust gene silencing mediated by antisense small RNAs in the pathogenic protist Entamoeba histolytica

    PubMed Central

    Morf, Laura; Pearson, Richard J.; Wang, Angelia S.; Singh, Upinder

    2013-01-01

    RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5′-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a ‘trigger’ and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5′-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects. PMID:23935116

  1. A Combinatorial Code for Splicing Silencing: UAGG and GGGG Motifs

    PubMed Central

    An, Ping; Burge, Christopher B

    2005-01-01

    Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5′-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes. PMID:15828859

  2. A combinatorial code for splicing silencing: UAGG and GGGG motifs.

    PubMed

    Han, Kyoungha; Yeo, Gene; An, Ping; Burge, Christopher B; Grabowski, Paula J

    2005-05-01

    Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes.

  3. Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila.

    PubMed

    Cernilogar, Filippo M; Onorati, Maria Cristina; Kothe, Greg O; Burroughs, A Maxwell; Parsi, Krishna Mohan; Breiling, Achim; Lo Sardo, Federica; Saxena, Alka; Miyoshi, Keita; Siomi, Haruhiko; Siomi, Mikiko C; Carninci, Piero; Gilmour, David S; Corona, Davide F V; Orlando, Valerio

    2011-11-06

    RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21-30 nucleotide) RNAs. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.

  4. Some Sources of Error in the Transcription of Real Time in Spoken Discourse.

    ERIC Educational Resources Information Center

    O'Connell, Daniel C.; Kowal, Sabine

    1990-01-01

    Discusses such errors in transcribing real time in spoken discourse as inconsistent use of transcriptional conventions; use of transcriptional symbols with multiple meanings; measurement problems; some cross-purposes of real-time transcription; neglect of time between onset and offset of speech and silence transcription; and transcriptions that…

  5. Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing

    PubMed Central

    Adams, Keith L.; Cronn, Richard; Percifield, Ryan; Wendel, Jonathan F.

    2003-01-01

    Most eukaryotes have genomes that exhibit high levels of gene redundancy, much of which seems to have arisen from one or more cycles of genome doubling. Polyploidy has been particularly prominent during flowering plant evolution, yielding duplicated genes (homoeologs) whose expression may be retained or lost either as an immediate consequence of polyploidization or on an evolutionary timescale. Expression of 40 homoeologous gene pairs was assayed by cDNA-single-stranded conformation polymorphism in natural (1- to 2-million-yr-old) and synthetic tetraploid cotton (Gossypium) to determine whether homoeologous gene pairs are expressed at equal levels after polyploid formation. Silencing or unequal expression of one homoeolog was documented for 10 of 40 genes examined in ovules of Gossypium hirsutum. Assays of homoeolog expression in 10 organs revealed variable expression levels and silencing, depending on the gene and organ examined. Remarkably, silencing and biased expression of some gene pairs are reciprocal and developmentally regulated, with one homoeolog showing silencing in some organs and the other being silenced in other organs, suggesting rapid subfunctionalization. Duplicate gene expression was examined in additional natural polyploids to characterize the pace at which expression alteration evolves. Analysis of a synthetic tetraploid revealed homoeolog expression and silencing patterns that sometimes mirrored those of the natural tetraploid. Both long-term and immediate responses to polyploidization were implicated. Data suggest that some silencing events are epigenetically induced during the allopolyploidization process. PMID:12665616

  6. Mu killer-Mediated and Spontaneous Silencing of Zea mays Mutator Family Transposable Elements Define Distinctive Paths of Epigenetic Inactivation

    PubMed Central

    Skibbe, David S.; Fernandes, J. F.; Walbot, Virginia

    2012-01-01

    Mu killer contains a partial inverted duplication of the mudrA transposase gene and two copies of the terminal inverted repeat A (TIRA) region of the master MuDR element of maize. Mu killer can effectively silence single copy MuDR/Mu lines, and it is proposed that a ∼4 kb hairpin RNA is generated by read through transcription from a flanking gene and that this transcript serves as a substrate for siRNA production. Mu killer was sequenced, except for a recalcitrant portion in the center of the locus, and shown to be co-linear with mudrA as originally proposed. The ability of the dominant Mu killer locus to silence a standard high copy number MuDR/Mu transposon line was evaluated. After two generations of exposure, about three quarters of individuals were silenced indicating reasonable effectiveness as measured by the absence of mudrA transposase transcripts. Mu killer individuals that effectively silenced MuDR expressed two short antisense transcripts. In contrast, Mu killer individuals that failed to silence MuDR expressed multiple sense transcripts, derived from read through transcription initiating in a flanking gene, but no antisense transcripts were detected. PMID:22993515

  7. Fetal hemoglobin silencing in humans

    PubMed Central

    Oneal, Patricia A.; Gantt, Nicole M.; Schwartz, Joseph D.; Bhanu, Natarajan V.; Lee, Y. Terry; Moroney, John W.; Reed, Christopher H.; Schechter, Alan N.; Luban, Naomi L. C.; Miller, Jeffery L.

    2006-01-01

    Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a “switching” model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies. PMID:16735596

  8. Breaking the Code of Silence.

    ERIC Educational Resources Information Center

    Halbig, Wolfgang W.

    2000-01-01

    Schools and communities must break the adolescent code of silence concerning threats of violence. Schools need character education stressing courage, caring, and responsibility; regular discussions of the school discipline code; formal security discussions with parents; 24-hour hotlines; and protocols for handling reports of potential violence.…

  9. Silence or the Sound of Limpid Water: Disability, Power, and the Educationalisation of Silence

    ERIC Educational Resources Information Center

    Verstraete, Pieter

    2017-01-01

    In this article the history of silence is looked at from an educational perspective. By closely examining the way three nineteenth-century authors--who all based their educational theories on concrete experiences with persons with disabilities--have related themselves to silence, it will be argued that silence has been educationalised. Silence has…

  10. Silence as the Foundation of Learning

    ERIC Educational Resources Information Center

    Caranfa, Angelo

    2004-01-01

    Past and present discussions on education all too frequently neglect the role that silence plays in learning. In this article I set out to demonstrate that silence is the very foundation of learning. My claim is that we must find ways of freeing silence in our pedagogical practices so that our discourse does not denigrate into mere empty words,…

  11. Conifers have a unique small RNA silencing signature

    PubMed Central

    Dolgosheina, Elena V.; Morin, Ryan D.; Aksay, Gozde; Sahinalp, S. Cenk; Magrini, Vincent; Mardis, Elaine R.; Mattsson, Jim; Unrau, Peter J.

    2008-01-01

    Plants produce small RNAs to negatively regulate genes, viral nucleic acids, and repetitive elements at either the transcriptional or post-transcriptional level in a process that is referred to as RNA silencing. While RNA silencing has been extensively studied across the different phyla of the animal kingdom (e.g., mouse, fly, worm), similar studies in the plant kingdom have focused primarily on angiosperms, thus limiting evolutionary studies of RNA silencing in plants. Here we report on an unexpected phylogenetic difference in the size distribution of small RNAs among the vascular plants. By extracting total RNA from freshly growing shoot tissue, we conducted a survey of small RNAs in 24 vascular plant species. We find that conifers, which radiated from the other seed-bearing plants ∼260 million years ago, fail to produce significant amounts of 24-nucleotide (nt) RNAs that are known to guide DNA methylation and heterochromatin formation in angiosperms. Instead, they synthesize a diverse population of small RNAs that are exactly 21-nt long. This finding was confirmed by high-throughput sequencing of the small RNA sequences from a conifer, Pinus contorta. A conifer EST search revealed the presence of a novel Dicer-like (DCL) family, which may be responsible for the observed change in small RNA expression. No evidence for DCL3, an enzyme that matures 24-nt RNAs in angiosperms, was found. We hypothesize that the diverse class of 21-nt RNAs found in conifers may help to maintain organization of their unusually large genomes. PMID:18566193

  12. Accumulation of RNA on chromatin disrupts heterochromatic silencing.

    PubMed

    Brönner, Cornelia; Salvi, Luca; Zocco, Manuel; Ugolini, Ilaria; Halic, Mario

    2017-04-12

    Long non-coding RNAs (lncRNAs) play a conserved role in regulating gene expression, chromatin dynamics and cell differentiation. They serve as a platform for RNA interference (RNAi)-mediated heterochromatin formation or DNA methylation in many eukaryotic organisms. We found in Schizosaccharomyces pombe, that heterochromatin is lost at transcribed regions in absence of RNA degradation. We show that heterochromatic RNAs are retained on chromatin, form DNA:RNA hybrids and need to be degraded by the Ccr4-Not complex or RNAi to maintain heterochromatic silencing. The Ccr4-Not complex is localized to chromatin independently of H3K9me and degrades chromatin associated transcripts, which is required for transcriptional silencing. Overexpression of heterochromatic RNA, but not euchromatic RNA, leads to its chromatin localization and loss of silencing of a distant ade6 reporter in wild type cells. Our results demonstrate that chromatin bound RNAs disrupt heterochromatin organization and need to be degraded in a process of heterochromatin formation.

  13. Is silence killing your company?

    PubMed

    Perlow, Leslie; Williams, Stephanie

    2003-05-01

    Many times, often with the best of intentions, people at work decide it's more productive to remain silent about their differences than to air them. There's no time, they think, or no point in going against what the boss says. But as new research by the authors shows, silencing doesn't smooth things over or make people more productive. It merely pushes differences beneath the surface and can set in motion powerfully destructive forces. When people stay silent about important disagreements, they can begin to fill with anxiety, anger, and resentment. As long as the conflict is unresolved, their repressed feelings remain potent, making them increasingly distrustful, self-protective, and all the more fearful that if they speak up they will be embarrassed or rejected. Their sense of insecurity grows, leading to further acts of silence, more defensiveness, and more distrust, thereby setting into motion a destructive "spiral of silence." Sooner or later, they mentally opt out--sometimes merely doing what they're told but contributing nothing of their own, sometimes spreading discontent and frustration throughout the workplace that can lead them, and others, to leave without thinking it through. These vicious spirals of silence can be replaced with virtuous spirals of communication, but that requires individuals to find the courage to act differently and executives to create the conditions in which people will value the expression of differences. All too often, behind failed products, broken processes, and mistaken career decisions are people who chose to hold their tongues. Breaking the silence can bring an outpouring of fresh ideas from all levels of an organization--ideas that might just raise the organization's performance to a whole new level.

  14. Polycomb Group-Dependent, Heterochromatin Protein 1-Independent, Chromatin Structures Silence Retrotransposons in Somatic Tissues Outside Ovaries

    PubMed Central

    Dufourt, J.; Brasset, E.; Desset, S.; Pouchin, P.; Vaury, C.

    2011-01-01

    Somatic cells are equipped with different silencing mechanisms that protect the genome against retrotransposons. In Drosophila melanogaster, a silencing pathway implicating the argonaute protein PIWI represses retrotransposons in cells surrounding the oocyte, whereas a PIWI-independent pathway is involved in other somatic tissues. Here, we show that these two silencing mechanisms result in distinct chromatin structures. Using sensor transgenes, we found that, in somatic tissues outside of the ovaries, these transgenes adopt a heterochromatic configuration implicating hypermethylation of H3K9 and K27. We identified the Polycomb repressive complexes (PRC1 and 2), but not heterochromatin protein 1 to be necessary factors for silencing. Once established, the compact structure is stably maintained through cell divisions. By contrast, in cells where the silencing is PIWI-dependent, the transgenes display an open and labile chromatin structure. Our data suggest that a post-transcriptional gene silencing (PTGS) mechanism is responsible for the repression in the ovarian somatic cells, whereas a mechanism that couples PTGS to transcriptional gene silencing operates to silence retrotransposons in the other somatic tissues. PMID:21908513

  15. Strategies for silencing and escape: the ancient struggle between transposable elements and their hosts.

    PubMed

    Lisch, Damon; Slotkin, R Keith

    2011-01-01

    Over the past several years, there has been an explosion in our understanding of the mechanisms by which plant transposable elements (TEs) are epigenetically silenced and maintained in an inactive state over long periods of time. This highly efficient process results in vast numbers of inactive TEs; indeed, the majority of many plant genomes are composed of these quiescent elements. This observation has led to the rather static view that TEs represent an essentially inert portion of plant genomes. However, recent work has demonstrated that TE silencing is a highly dynamic process that often involves transcription of TEs at particular times and places during plant development. Plants appear to use transcripts from silenced TEs as an ongoing source of information concerning the mobile portion of the genome. In contrast to our understanding of silencing pathways, we know relatively little about the ways in which TEs evade silencing. However, vast differences in TE content between even closely related plant species suggest that they are often wildly successful at doing so. Here, we discuss TE activity in plants as the result of a constantly shifting balance between host strategies for TE silencing and TE strategies for escape and amplification.

  16. Two Components of the RNA-Directed DNA Methylation Pathway Associate with MORC6 and Silence Loci Targeted by MORC6 in Arabidopsis

    PubMed Central

    Liu, Zhang-Wei; Zhou, Jin-Xing; Huang, Huan-Wei; Li, Yong-Qiang; Shao, Chang-Rong; Li, Lin; Cai, Tao; Chen, She

    2016-01-01

    The SU(VAR)3-9 homolog SUVH9 and the double-stranded RNA-binding protein IDN2 were thought to be components of an RNA-directed DNA methylation (RdDM) pathway in Arabidopsis. We previously found that SUVH9 interacts with MORC6 but how the interaction contributes to transcriptional silencing remains elusive. Here, our genetic analysis indicates that SUVH2 and SUVH9 can either act in the same pathway as MORC6 or act synergistically with MORC6 to mediate transcriptional silencing. Moreover, we demonstrate that IDN2 interacts with MORC6 and mediates the silencing of a subset of MORC6 target loci. Like SUVH2, SUVH9, and IDN2, other RdDM components including Pol IV, Pol V, RDR2, and DRM2 are also required for transcriptional silencing at a subset of MORC6 target loci. MORC6 was previously shown to mediate transcriptional silencing through heterochromatin condensation. We demonstrate that the SWI/SNF chromatin-remodeling complex components SWI3B, SWI3C, and SWI3D interact with MORC6 as well as with SUVH9 and then mediate transcriptional silencing. These results suggest that the RdDM components are involved not only in DNA methylation but also in MORC6-mediated heterochromatin condensation. This study illustrates how DNA methylation is linked to heterochromatin condensation and thereby enhances transcriptional silencing at methylated genomic regions. PMID:27171427

  17. Cancer-associated TERT promoter mutations abrogate telomerase silencing.

    PubMed

    Chiba, Kunitoshi; Johnson, Joshua Z; Vogan, Jacob M; Wagner, Tina; Boyle, John M; Hockemeyer, Dirk

    2015-07-21

    Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells.

  18. RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops.

    PubMed

    Guo, Qigao; Liu, Qing; Smith, Neil A; Liang, Guolu; Wang, Ming-Bo

    2016-12-01

    Understanding the fundamental nature of a molecular process or a biological pathway is often a catalyst for the development of new technologies in biology. Indeed, studies from late 1990s to early 2000s have uncovered multiple overlapping but functionally distinct RNA silencing pathways in plants, including the posttranscriptional microRNA and small interfering RNA pathways and the transcriptional RNA-directed DNA methylation pathway. These findings have in turn been exploited for developing artificial RNA silencing technologies such as hairpin RNA, artificial microRNA, intrinsic direct repeat, 3' UTR inverted repeat, artificial trans-acting siRNA, and virus-induced gene silencing technologies. Some of these RNA silencing technologies, such as the hairpin RNA technology, have already been widely used for genetic improvement of crop plants in agriculture. For horticultural plants, RNA silencing technologies have been used to increase disease and pest resistance, alter plant architecture and flowering time, improve commercial traits of fruits and flowers, enhance nutritional values, remove toxic compounds and allergens, and develop high-value industrial products. In this article we aim to provide an overview of the RNA silencing pathways in plants, summarize the existing RNA silencing technologies, and review the current progress in applying these technologies for the improvement of agricultural crops particularly horticultural crops.

  19. RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops

    PubMed Central

    Guo, Qigao; Liu, Qing; Smith, Neil A.; Liang, Guolu; Wang, Ming-Bo

    2016-01-01

    Understanding the fundamental nature of a molecular process or a biological pathway is often a catalyst for the development of new technologies in biology. Indeed, studies from late 1990s to early 2000s have uncovered multiple overlapping but functionally distinct RNA silencing pathways in plants, including the posttranscriptional microRNA and small interfering RNA pathways and the transcriptional RNA-directed DNA methylation pathway. These findings have in turn been exploited for developing artificial RNA silencing technologies such as hairpin RNA, artificial microRNA, intrinsic direct repeat, 3’ UTR inverted repeat, artificial trans-acting siRNA, and virus-induced gene silencing technologies. Some of these RNA silencing technologies, such as the hairpin RNA technology, have already been widely used for genetic improvement of crop plants in agriculture. For horticultural plants, RNA silencing technologies have been used to increase disease and pest resistance, alter plant architecture and flowering time, improve commercial traits of fruits and flowers, enhance nutritional values, remove toxic compounds and allergens, and develop high-value industrial products. In this article we aim to provide an overview of the RNA silencing pathways in plants, summarize the existing RNA silencing technologies, and review the current progress in applying these technologies for the improvement of agricultural crops particularly horticultural crops. PMID:28217004

  20. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA.

    PubMed

    Decker, Logan M; Xiao, Hua; Boone, Erin C; Vierling, Michael M; Shanker, Benjamin S; Kingston, Shanika L; Boone, Shannon F; Haynes, Jackson B; Shiu, Patrick K T

    2017-02-07

    In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus to combat these repetitive elements. One of these defense mechanisms is known as meiotic silencing by unpaired DNA (MSUD), which is an RNA silencing system that identifies and silences unpaired genes during meiosis. Utilizing common RNAi proteins such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified another silencing component known as the cap-binding complex (CBC). Made up of CBP20 and CBP80 (cap-binding proteins 20 and 80), CBC associates with the 5' cap of nascent mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. Similar to animals but unlike plants, CBP20's robust nuclear re-entry is shown to be dependent on CBP80. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors.

  1. Adult-specific electrical silencing of pacemaker neurons uncouples the molecular oscillator from circadian outputs

    PubMed Central

    Depetris-Chauvin, Ana; Berni, Jimena; Aranovich, Ezequiel J.; Muraro, Nara I.; Beckwith, Esteban J.; Ceriani, María Fernanda

    2011-01-01

    Summary Background Circadian rhythms regulate physiology and behavior through transcriptional feedback loops of clock genes running within specific pacemaker cells. In Drosophila, molecular oscillations in the small ventral Lateral Neurons (sLNvs) command rhythmic behavior under free-running conditions releasing the neuropeptide PIGMENT DISPERSING FACTOR (PDF) in a circadian fashion. Electrical activity in the sLNvs is also required for behavioral rhythmicity. Yet, how temporal information is transduced into behavior remains unclear. Results Here we developed a new tool for temporal control of gene expression to obtain adult-restricted electrical silencing of the PDF circuit, which led to reversible behavioral arrhythmicity. Remarkably, PER oscillations during the silenced phase remained unaltered, indicating that arrhythmicity is a direct consequence of the silenced activity. Accordingly, circadian axonal remodeling and PDF accumulation were severely affected during the silenced phase. Conclusions Although electrical activity of the sLNvs is not a clock component it coordinates circuit outputs leading to rhythmic behavior. PMID:22018542

  2. Two suppressors of RNA silencing encoded by cereal-infecting members of the family Luteoviridae.

    PubMed

    Liu, Yan; Zhai, Hao; Zhao, Kun; Wu, Beilei; Wang, Xifeng

    2012-08-01

    Several members of the family Luteoviridae are important pathogens of cultivated plant species of the family Gramineae. In this study, we explored RNA-silencing suppressors (RSSs) encoded by two cereal-infecting luteoviruses: barley yellow dwarf virus and wheat yellow dwarf virus (BYDV and WYDV, respectively). The P0 protein of WYDV-GPV (P0(GPV)) and the P6 protein of BYDV-GAV (P6(GAV)) displayed RSS activities when expressed in agro-infiltrated leaves of Nicotiana benthamiana, by their local ability to inhibit post-transcriptional gene silencing of GFP. Analysis of GFP, mRNA and GFP-specific small interfering RNA indicated that both P0(GPV) and P6(GAV) are suppressors of silencing that can restrain not only local but also systemic gene silencing. This is the first report of RSS activity of the P6 protein in a member of the genus Luteovirus.

  3. H2A.Z Maintenance During Mitosis Reveals Nucleosome Shifting on Mitotically Silenced Genes

    PubMed Central

    Kelly, Theresa K.; Miranda, Tina Branscombe; Liang, Gangning; Berman, Benjamin P.; Lin, Joy C.; Tanay, Amos; Jones, Peter A.

    2010-01-01

    Profound chromatin changes occur during mitosis to allow for gene silencing and chromosome segregation followed by re-activation of memorized transcription states in daughter cells. Using genome-wide sequencing, we found H2A.Z containing +1 nucleosomes of active genes shift upstream to occupy TSSs during mitosis, significantly reducing nucleosome-depleted regions. Single molecule analysis confirmed nucleosome shifting and demonstrated that mitotic shifting is specific to active genes that are silenced during mitosis and thus is not seen on promoters, which are silenced by methylation or mitotically expressed genes. Using the GRP78 promoter as a model, we found H3K4 tri-methylation is also maintained while other indicators of active chromatin are lost and expression is decreased. These key changes provide a potential mechanism for rapid silencing and re-activation of genes during the cell cycle. PMID:20864037

  4. RNA Pol IV and V in Gene Silencing: Rebel Polymerases Evolving Away From Pol II’s Rules

    PubMed Central

    Zhou, Ming; Law, Julie A.

    2015-01-01

    Noncoding RNAs regulate gene expression at both the transcriptional and post-transcriptional levels, and play critical roles in development, imprinting and the maintenance of genome integrity in eukaryotic organisms [1–3]. Therefore, it is important to understand how the production of such RNAs are controlled. In addition to the three canonical DNA dependent RNA polymerases (Pol) Pol I, II and III, two non-redundant plant-specific RNA polymerases, Pol IV and Pol V, have been identified and shown to generate noncoding RNAs that are required for transcriptional gene silencing via the RNA-directed DNA methylation (RdDM) pathway. Thus, somewhat paradoxically, transcription is required for gene silencing. This paradox extends beyond plants, as silencing pathways in yeast, fungi, flies, worms, and mammals also require transcriptional machinery [4,5]. As plants have evolved specialized RNA polymerases to carry out gene silencing in a manner that is separate from the essential roles of Pol II, their characterization offers unique insight into how RNA polymerases facilitate gene silencing. In this review, we focus on the mechanisms of Pol IV and Pol V function, including their compositions, their transcripts, and their modes of recruitment to chromatin. PMID:26344361

  5. RNA silencing and plant viral diseases.

    PubMed

    Wang, Ming-Bo; Masuta, Chikara; Smith, Neil A; Shimura, Hanako

    2012-10-01

    RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.

  6. Contribution of transcription to animal early development.

    PubMed

    Wang, Jianbin; Davis, Richard E

    2014-01-01

    In mature gametes and during the oocyte-to-embryo transition, transcription is generally silenced and gene expression is post-transcriptionally regulated. However, we recently discovered that major transcription can occur immediately after fertilization, prior to pronuclear fusion, and in the first cell division of the oocyte-to-embryo transition in the nematode Ascaris suum. We postulate that the balance between transcriptional and post-transcriptional regulation during the oocyte-to-embryo transition may largely be determined by cell cycle length and thus the time available for the genome to be transcribed.

  7. Light intensity and temperature affect systemic spread of silencing signal in transient agroinfiltration studies.

    PubMed

    Patil, Basavaprabhu L; Fauquet, Claude M

    2015-06-01

    RNA silencing is a sequence-specific post-transcriptional gene inactivation mechanism that operates in diverse organisms and that can extend beyond its site of initiation, owing to the movement of the silencing signal, called non-autonomous gene silencing. Previous studies have shown that several factors manifest the movement of the silencing signal, such as the size (21 or 24 nucleotides) of the secondary small interfering RNA (siRNA) produced, the steady-state concentration of siRNAs and their cognate messenger RNA (mRNA) or a change in the sink-source status of plant parts affecting phloem translocation. Our study shows that both light intensity and temperature have a significant impact on the systemic movement of the silencing signal in transient agroinfiltration studies in Nicotiana benthamiana. At higher light intensities (≥ 450 μE/m(2)/s) and higher temperatures (≥ 30 °C), gene silencing was localized to leaf tissue that was infiltrated, without any systemic spread. Interestingly, in these light and temperature conditions (≥ 450 μE/m(2) /s and ≥ 30 °C), the N. benthamiana plants showed recovery from the viral symptoms. However, the reduced systemic silencing and reduced viral symptom severity at higher light intensities were caused by a change in the sink-source status of the plant, ultimately affecting the phloem translocation of small RNAs or the viral genome. In contrast, at lower light intensities (<300 μE/m(2)/s) with a constant temperature of 25 °C, there was strong systemic movement of the silencing signal in the N. benthamiana plants and reduced recovery from virus infections. The accumulation of gene-specific siRNAs was reduced at higher temperature as a result of a reduction in the accumulation of transcript on transient agroinfiltration of RNA interference (RNAi) constructs, mostly because of poor T-DNA transfer activity of Agrobacterium, possibly also accompanied by reduced phloem translocation.

  8. Nickel and Epigenetic Gene Silencing

    PubMed Central

    Sun, Hong; Shamy, Magdy; Costa, Max

    2013-01-01

    Insoluble nickel compounds are well-established human carcinogens. Occupational exposure to these compounds leads to increased incidence of lung and nasal cancer in nickel refinery workers. Apart from its weak mutagenic activity and hypoxia mimicking effect there is mounting experimental evidence indicating that epigenetic alteration plays an important role in nickel-induced carcinogenesis. Multiple epigenetic mechanisms have been identified to mediate nickel-induced gene silencing. Nickel ion is able to induce heterochromatinization by binding to DNA-histone complexes and initiating chromatin condensation. The enzymes required for establishing or removing epigenetic marks can be targeted by nickel, leading to altered DNA methylation and histone modification landscapes. The current review will focus on the epigenetic changes that contribute to nickel-induced gene silencing. PMID:24705264

  9. Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis.

    PubMed

    Xiao, Jun; Jin, Run; Yu, Xiang; Shen, Max; Wagner, John D; Pai, Armaan; Song, Claire; Zhuang, Michael; Klasfeld, Samantha; He, Chongsheng; Santos, Alexandre M; Helliwell, Chris; Pruneda-Paz, Jose L; Kay, Steve A; Lin, Xiaowei; Cui, Sujuan; Garcia, Meilin Fernandez; Clarenz, Oliver; Goodrich, Justin; Zhang, Xiaoyu; Austin, Ryan S; Bonasio, Roberto; Wagner, Doris

    2017-08-21

    Disruption of gene silencing by Polycomb protein complexes leads to homeotic transformations and altered developmental-phase identity in plants. Here we define short genomic fragments, known as Polycomb response elements (PREs), that direct Polycomb repressive complex 2 (PRC2) placement at developmental genes regulated by silencing in Arabidopsis thaliana. We identify transcription factor families that bind to these PREs, colocalize with PRC2 on chromatin, physically interact with and recruit PRC2, and are required for PRC2-mediated gene silencing in vivo. Two of the cis sequence motifs enriched in the PREs are cognate binding sites for the identified transcription factors and are necessary and sufficient for PRE activity. Thus PRC2 recruitment in Arabidopsis relies in large part on binding of trans-acting factors to cis-localized DNA sequence motifs.

  10. HMGB1 silence could promote MCF-7 cell apoptosis and inhibit invasion and metastasis

    PubMed Central

    Ni, Ping; Zhang, Yongjian; Liu, Yueqin; Lin, Xin; Su, Xiaolian; Lu, Hongxiang; Shen, Huiling; Xu, Wenlin; Xu, Huaxi; Su, Zhaoliang

    2015-01-01

    High mobility group box 1 (HMGB1), a non-histone nuclear protein, was associated with a variety of biological important processes, such as transcription, differentiation, extracellular signaling. As a cytokine or inflammatory mediator, more and more data showed that HMGB1 was involved in inflammatory diseases, cancers or autoimmune disease. However, few data focused on nucleic or cytoplasmic function of HMGB1. Therefore, the present study focused on cancer cells biological characteristics following HMGB1 silence. HMGB1 siRNAs were designed and chemically synthesized, and then transfected into the breast cancer cell line MCF-7 with lipofectamine 2000. The transcription and translation level of HMGB1 expression, proliferation, apoptosis, migration of MCF-7 were determined. The results demonstrated that HMGB1 silence inhibit invasion and migration and promote apoptosis of human breast cells; which indicated that HMGB1 silence might be a potential therapy targets. PMID:26884867

  11. HMGB1 silence could promote MCF-7 cell apoptosis and inhibit invasion and metastasis.

    PubMed

    Ni, Ping; Zhang, Yongjian; Liu, Yueqin; Lin, Xin; Su, Xiaolian; Lu, Hongxiang; Shen, Huiling; Xu, Wenlin; Xu, Huaxi; Su, Zhaoliang

    2015-01-01

    High mobility group box 1 (HMGB1), a non-histone nuclear protein, was associated with a variety of biological important processes, such as transcription, differentiation, extracellular signaling. As a cytokine or inflammatory mediator, more and more data showed that HMGB1 was involved in inflammatory diseases, cancers or autoimmune disease. However, few data focused on nucleic or cytoplasmic function of HMGB1. Therefore, the present study focused on cancer cells biological characteristics following HMGB1 silence. HMGB1 siRNAs were designed and chemically synthesized, and then transfected into the breast cancer cell line MCF-7 with lipofectamine 2000. The transcription and translation level of HMGB1 expression, proliferation, apoptosis, migration of MCF-7 were determined. The results demonstrated that HMGB1 silence inhibit invasion and migration and promote apoptosis of human breast cells; which indicated that HMGB1 silence might be a potential therapy targets.

  12. Eos mediates Foxp3-dependent gene silencing in CD4+ regulatory T cells.

    PubMed

    Pan, Fan; Yu, Hong; Dang, Eric V; Barbi, Joseph; Pan, Xiaoyu; Grosso, Joseph F; Jinasena, Dinili; Sharma, Sudarshana M; McCadden, Erin M; Getnet, Derese; Drake, Charles G; Liu, Jun O; Ostrowski, Michael C; Pardoll, Drew M

    2009-08-28

    CD4+ regulatory T cells (Tregs) maintain immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses. The core genetic program of Tregs and their ability to suppress pathologic immune responses depends on the transcription factor Foxp3. Despite progress in understanding mechanisms of Foxp3-dependent gene activation, the molecular mechanism of Foxp3-dependent gene repression remains largely unknown. We identified Eos, a zinc-finger transcription factor of the Ikaros family, as a critical mediator of Foxp3-dependent gene silencing in Tregs. Eos interacts directly with Foxp3 and induces chromatin modifications that result in gene silencing in Tregs. Silencing of Eos in Tregs abrogates their ability to suppress immune responses and endows them with partial effector function, thus demonstrating the critical role that Eos plays in Treg programming.

  13. A Gli silencer is required for robust repression of gremlin in the vertebrate limb bud.

    PubMed

    Li, Qiang; Lewandowski, Jordan P; Powell, Marian B; Norrie, Jacqueline L; Cho, Seung Hee; Vokes, Steven A

    2014-05-01

    The transcriptional response to the Hedgehog (Hh) pathway is mediated by Gli proteins, which function as context-dependent transcriptional activators or repressors. However, the mechanism by which Gli proteins regulate their target genes is poorly understood. Here, we have performed the first genetic characterization of a Gli-dependent cis-regulatory module (CRM), focusing on its regulation of Grem1 in the mouse limb bud. The CRM, termed GRE1 (Gli responsive element 1), can act as both an enhancer and a silencer. The enhancer activity requires sustained Hh signaling. As a Gli-dependent silencer, GRE1 prevents ectopic transcription of Grem1 driven through additional CRMs. In doing so, GRE1 works with additional GREs to robustly regulate Grem1. We suggest that multiple Gli CRMs may be a general mechanism for mediating a robust transcriptional response to the Hh pathway.

  14. Sex-induced silencing defends the genome of Cryptococcus neoformans via RNAi

    PubMed Central

    Wang, Xuying; Hsueh, Yen-Ping; Li, Wenjun; Floyd, Anna; Skalsky, Rebecca; Heitman, Joseph

    2010-01-01

    Cosuppression is a silencing phenomenon triggered by the introduction of homologous DNA sequences into the genomes of organisms as diverse as plants, fungi, flies, and nematodes. Here we report sex-induced silencing (SIS), which is triggered by tandem integration of a transgene array in the human fungal pathogen Cryptococcus neoformans. A SXI2a-URA5 transgene array was found to be post-transcriptionally silenced during sexual reproduction. More than half of the progeny that inherited the SXI2a-URA5 transgene became uracil-auxotrophic due to silencing of the URA5 gene. In vegetative mitotic growth, silencing of this transgene array occurred at an ∼250-fold lower frequency, indicating that silencing is induced during the sexual cycle. Central components of the RNAi pathway—including genes encoding Argonaute, Dicer, and an RNA-dependent RNA polymerase—are all required for both meiotic and mitotic transgene silencing. URA5-derived ∼22-nucleotide (nt) small RNAs accumulated in the silenced isolates, suggesting that SIS is mediated by RNAi via sequence-specific small RNAs. Through deep sequencing of the small RNA population in C. neoformans, we also identified abundant small RNAs mapping to repetitive transposable elements, and these small RNAs were absent in rdp1 mutant strains. Furthermore, a group of retrotransposons was highly expressed during mating of rdp1 mutant strains, and an increased transposition/mutation rate was detected in their progeny, indicating that the RNAi pathway squelches transposon activity during the sexual cycle. Interestingly, Ago1, Dcr1, Dcr2, and Rdp1 are translationally induced in mating cells, and Ago1, Dcr1, and Dcr2 localize to processing bodies (P bodies), whereas Rdp1 appears to be nuclear, providing mechanistic insights into the elevated silencing efficiency during sexual reproduction. We hypothesize that the SIS RNAi pathway operates to defend the genome during sexual development. PMID:21078820

  15. Limitations of silencing at native yeast telomeres.

    PubMed Central

    Pryde, F E; Louis, E J

    1999-01-01

    Silencing at native yeast telomeres, in which the subtelomeric elements are intact, is different from silencing at terminal truncations. The repression of URA3 inserted in different subtelomeric positions at several chromosome ends was investigated. Many ends exhibit very little silencing close to the telomere, while others exhibit substantial repression in limited domains. Silencing at native ends is discontinuous, with maximal repression found adjacent to the ARS consensus sequence in the subtelomeric core X element. The level of repression declines precipitously towards the centromere. Mutation of the ARS sequence or an adjacent Abf1p-binding site significantly reduces silencing. The subtelomeric Y' elements are resistant to silencing along their whole length, yet silencing can be re-established at the proximal X element. Deletion of PPR1, the transactivator of URA3, and SIR3 overexpression do not increase repression or extend spreading of silencing to the same extent as with terminally truncated ends. sir1Delta causes partial derepression at X-ACS, in contrast to the lack of effect seen at terminal truncations. orc2-1 and orc5-1 have no effect on natural silencing yet cause derepression at truncated ends. X-ACS silencing requires the proximity of the telomere and is dependent on SIR2, SIR3, SIR4 and HDF1. The structures found at native yeast telomeres appear to limit the potential of repressive chromatin. PMID:10228167

  16. Gene Silencing in Adult Aedes aegypti Mosquitoes Through Oral Delivery of Double-Stranded RNA

    DTIC Science & Technology

    2012-01-01

    inspired by the success of Remebee in targeting the virus impli- cated in colony collapse disorder in honeybees (Maori et al. 2009; Hunter et al. 2010...invasive method for silencing gene transcription in honeybees maintained under natural conditions. Insect Biochem. Mol. Biol. 39, 157–160. Patrick ML

  17. Silencing mechansim of C5 transgenic plums is stable under challenge inoculation with heterologous viruses

    USDA-ARS?s Scientific Manuscript database

    Transgenic C5 'HoneySweet' is a clone of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP). This transgenic plum displays post-transcriptional gene silencing (PTGS) which makes it highly resistant to PPV infection. To test the effect of heterologous viruses on the ...

  18. Silencing honey bee naked cuticle (nkd) reduces Nosema ceranae replication and disease levels

    USDA-ARS?s Scientific Manuscript database

    Nosema ceranae is a new and emerging microsporidian parasite of European honey bees, Apis mellifera that has been implicated in alarming colony losses worldwide. RNA interference (RNAi), a post-transcriptional gene silencing mechanism, has emerged as a potent and specific strategy for controlling in...

  19. Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons

    PubMed Central

    Shpiz, Sergey; Olovnikov, Ivan; Sergeeva, Anna; Lavrov, Sergey; Abramov, Yuri; Savitsky, Mikhail; Kalmykova, Alla

    2011-01-01

    In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. Telomeric retroelements HeT-A, TART and TAHRE, which are involved in telomere maintenance in Drosophila, are also the targets of piRNA-mediated silencing. We have demonstrated that expression of reporter genes driven by the HeT-A promoter is under the control of the piRNA silencing pathway independent of the transgene location. In order to test directly whether piRNAs affect the transcriptional state of retrotransposons we performed a nuclear run-on (NRO) assay and revealed increased density of the active RNA polymerase complexes at the sequences of endogenous HeT-A and TART telomeric retroelements as well as HeT-A-containing constructs in the ovaries of spn-E mutants and in flies with piwi knockdown. This strongly correlates with enrichment of two histone H3 modifications (dimethylation of lysine 79 and dimethylation of lysine 4), which mark transcriptionally active chromatin, on the same sequences in the piRNA pathway mutants. spn-E mutation and piwi knockdown results in transcriptional activation of some other non-telomeric retrotransposons in the ovaries, such as I-element and HMS Beagle. Therefore piRNA-mediated transcriptional mode of silencing is involved in the control of retrotransposon expression in the Drosophila germline. PMID:21764773

  20. Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons.

    PubMed

    Shpiz, Sergey; Olovnikov, Ivan; Sergeeva, Anna; Lavrov, Sergey; Abramov, Yuri; Savitsky, Mikhail; Kalmykova, Alla

    2011-11-01

    In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. Telomeric retroelements HeT-A, TART and TAHRE, which are involved in telomere maintenance in Drosophila, are also the targets of piRNA-mediated silencing. We have demonstrated that expression of reporter genes driven by the HeT-A promoter is under the control of the piRNA silencing pathway independent of the transgene location. In order to test directly whether piRNAs affect the transcriptional state of retrotransposons we performed a nuclear run-on (NRO) assay and revealed increased density of the active RNA polymerase complexes at the sequences of endogenous HeT-A and TART telomeric retroelements as well as HeT-A-containing constructs in the ovaries of spn-E mutants and in flies with piwi knockdown. This strongly correlates with enrichment of two histone H3 modifications (dimethylation of lysine 79 and dimethylation of lysine 4), which mark transcriptionally active chromatin, on the same sequences in the piRNA pathway mutants. spn-E mutation and piwi knockdown results in transcriptional activation of some other non-telomeric retrotransposons in the ovaries, such as I-element and HMS Beagle. Therefore piRNA-mediated transcriptional mode of silencing is involved in the control of retrotransposon expression in the Drosophila germline.

  1. Tritimovirus P1 functions as a suppressor of RNA silencing and an enhancer of disease symptoms

    USDA-ARS?s Scientific Manuscript database

    Wheat streak mosaic virus (WSMV) is an eriophyid mite-transmitted virus of the genus Tritimovirus, family Potyviridae. Complete deletion of HC-Pro has no effect on WSMV virulence or disease synergism, suggesting that a different viral protein suppresses post-transcriptional gene silencing (PTGS). PT...

  2. Artificial micro RNA (amiRNA) induced gene silencing in alfalfa (Medicago sativa)

    USDA-ARS?s Scientific Manuscript database

    Gene silencing is a powerful technique that allows the study of the function of specific genes by selectively reducing their transcription. Several different approaches can be used; however, they all have in common the artificial generation of single-stranded small RNAs that are utilized by the endo...

  3. Berkson's bias, selection bias, and missing data.

    PubMed

    Westreich, Daniel

    2012-01-01

    Although Berkson's bias is widely recognized in the epidemiologic literature, it remains underappreciated as a model of both selection bias and bias due to missing data. Simple causal diagrams and 2 × 2 tables illustrate how Berkson's bias connects to collider bias and selection bias more generally, and show the strong analogies between Berksonian selection bias and bias due to missing data. In some situations, considerations of whether data are missing at random or missing not at random are less important than the causal structure of the missing data process. Although dealing with missing data always relies on strong assumptions about unobserved variables, the intuitions built with simple examples can provide a better understanding of approaches to missing data in real-world situations.

  4. Stress-Induced Activation of Heterochromatic Transcription

    PubMed Central

    Tittel-Elmer, Mireille; Bucher, Etienne; Broger, Larissa; Mathieu, Olivier; Paszkowski, Jerzy; Vaillant, Isabelle

    2010-01-01

    Constitutive heterochromatin comprising the centromeric and telomeric parts of chromosomes includes DNA marked by high levels of methylation associated with histones modified by repressive marks. These epigenetic modifications silence transcription and ensure stable inheritance of this inert state. Although environmental cues can alter epigenetic marks and lead to modulation of the transcription of genes located in euchromatic parts of the chromosomes, there is no evidence that external stimuli can globally destabilize silencing of constitutive heterochromatin. We have found that heterochromatin-associated silencing in Arabidopsis plants subjected to a particular temperature regime is released in a genome-wide manner. This occurs without alteration of repressive epigenetic modifications and does not involve common epigenetic mechanisms. Such induced release of silencing is mostly transient, and rapid restoration of the silent state occurs without the involvement of factors known to be required for silencing initiation. Thus, our results reveal new regulatory aspects of transcriptional repression in constitutive heterochromatin and open up possibilities to identify the molecular mechanisms involved. PMID:21060865

  5. Identification of RNA silencing components in soybean and sorghum

    PubMed Central

    2014-01-01

    Background RNA silencing is a process triggered by 21–24 small RNAs to repress gene expression. Many organisms including plants use RNA silencing to regulate development and physiology, and to maintain genome stability. Plants possess two classes of small RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). The frameworks of miRNA and siRNA pathways have been established in the model plant, Arabidopsis thaliana (Arabidopsis). Results Here we report the identification of putative genes that are required for the generation and function of miRNAs and siRNAs in soybean and sorghum, based on knowledge obtained from Arabidopsis. The gene families, including DCL, HEN1, SE, HYL1, HST, RDR, NRPD1, NRPD2/NRPE2, NRPE1, and AGO, were analyzed for gene structures, phylogenetic relationships, and protein motifs. The gene expression was validated using RNA-seq, expressed sequence tags (EST), and reverse transcription PCR (RT-PCR). Conclusions The identification of these components could provide not only insight into RNA silencing mechanism in soybean and sorghum but also basis for further investigation. All data are available at http://sysbio.unl.edu/. PMID:24387046

  6. [E. M. Jellinek's silenced and silencing transgenerational story].

    PubMed

    Kelemen, Gábor; Márk, Mónika

    2013-01-01

    Jellinek is a kind of archetypal character for future generations in the field of addiction studies. His implosion in the arena of alcoholism around the age of 50 was an unexpected challenge to medical science. We know very little about his own role models giving an intellectual and moral compass to his pragmatic creativity. More than 30 years has passed since Jellinek's death when an American sociologist Ron Roizen started unearthing his silent story. Roizen discerned that there are a lot of unsaid and muted issues in his personal Hungarian past. Our paper, based on the authors' research in Hungarian archives and other sources reveals that not just Jellinek's personal but his transgenerational narrative has been not-yet-said. This silenced and silencing history appears an unfinished business of acculturation of the family, which started prior to four generations. Authors have been concluding that the issue of religious conversion is a critical point in the process of acculturation. They examine the counter move of loyalty to family values and driving force of assimilation making their story unspeakable.

  7. Silence Is Consent, or Curse Ye Meroz!

    ERIC Educational Resources Information Center

    Levin, Richard

    1997-01-01

    Examines assumptions of "oppositional" literary criticism, namely the assumption that older-style "objective" literary criticism must, in its political silence, be supportive of dominant ideologies. (TB)

  8. Silence

    NASA Astrophysics Data System (ADS)

    Cogswell, J.

    2011-06-01

    On the occasion of the International Year of Astronomy, I was commissioned to create a mural for the University of Michigan Department of Astronomy, responding to an array of scientific images based on astronomical research, with special focus on the work of University of Michigan astronomers carried out within the building. My paper illustrates the development of this and several subsequent projects, explaining the implications for my artistic practice of entering into this conversation with astronomers and their work.

  9. A position-dependent silencer plays a major role in repressing. alpha. -fetoprotein expression in human hepatoma

    SciTech Connect

    Nakabayashi, Hidekazu; Hashimoto, Tomoko; Miyao, Yasuyoshi; Tjong, Kuokoewang; Chan, J.; Tamaoki, Taiki )

    1991-12-01

    A large percentage of human hepatomas produce {alpha}-fetoprotein (AFP), but the levels of AFP expression vary greatly among hepatomas. To understand the molecular basis for this variation. The authors analyzed transcriptional regulatory activities associated with the 5{prime}-flanking region of the AFP gene in two human hepatoma cell lines, HuH-7 and huH-1/cl-2, which produce a high and a low level of AFP, respectively. They found that the low level of AFP production in huH-1/cl-2 is due to the action of at least two silencer regions located between the enhancer and the promoter of the AFP gene. In contrast, no silencer activity is expressed in HuH-7. They identified 5{prime}-CTTCATAACTAATACTT-3{prime} to be a core sequence responsible for the negative regulatory activity. This sequence is repeated four times in a strong, distal silencer region, Sd, whereas one copy is present in a weak, proximal silencer region, Sp. The silencer reduces transcriptional initiation by blocking enhancer activation of the AFP promoter in a position-dependent manner. The silencer functions in the presence of positive transcription factors and may play a key role in developmental repression as well as variable expression of the AFP gene in hepatomas.

  10. Histone modifications are associated with the persistence or silencing of vector-mediated transgene expression in vivo.

    PubMed

    Riu, Efren; Chen, Zhi-Ying; Xu, Hui; He, Chen-Yi; Kay, Mark A

    2007-07-01

    One of the major obstacles to success in non-viral gene therapy is transcriptional silencing of the DNA vector. The mechanisms underlying gene silencing/repression in mammalian cells are complex and remain unclear. Because changes in chromatin structure and, in particular, histone modifications are involved in transcriptional regulation of endogenous genes, we hypothesized that changes in the pattern of histone modifications were related to the observed transcriptional silencing of exogenous DNA vectors. We used antibodies against specific modified histones to perform chromatin immunoprecipitation (ChIP) analyses on liver lysates from mice transfected with two types of plasmids: (i) DNA minicircles (MCs) devoid of bacterial plasmid backbone DNA, which showed marked persistence of transgene expression, and (ii) their parental plasmids, which were silenced over time. Silencing of the transgene from the parental vectors was accompanied by an increase in heterochromatin-associated histone modifications and a decrease in modifications typically associated with euchromatin. Conversely, the pattern of histone modifications on the MC DNA was consistent with euchromatin. Our data indicates that (i) episomal vectors undergo chromatinization in vivo, and (ii) both persistence and silencing of transgene expression are associated with specific histone modifications.

  11. RNAi pathway genes are resistant to small RNA mediated gene silencing in the protozoan parasite Entamoeba histolytica.

    PubMed

    Pompey, Justine M; Morf, Laura; Singh, Upinder

    2014-01-01

    The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a "trigger" to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing.

  12. RNAi Pathway Genes Are Resistant to Small RNA Mediated Gene Silencing in the Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Pompey, Justine M.; Morf, Laura; Singh, Upinder

    2014-01-01

    The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a “trigger” to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing. PMID:25198343

  13. RNA Quality Control as a Key to Suppressing RNA Silencing of Endogenous Genes in Plants.

    PubMed

    Liu, Lin; Chen, Xuemei

    2016-06-06

    RNA quality control of endogenous RNAs is an integral part of eukaryotic gene expression and often relies on exonucleolytic degradation to eliminate dysfunctional transcripts. In parallel, exogenous and selected endogenous RNAs are degraded through RNA silencing, which is a genome defense mechanism used by many eukaryotes. In plants, RNA silencing is triggered by the production of double-stranded RNAs (dsRNAs) by RNA-DEPENDENT RNA POLYMERASEs (RDRs) and proceeds through small interfering (si) RNA-directed, ARGONAUTE (AGO)-mediated cleavage of homologous transcripts. Many studies revealed that plants avert inappropriate posttranscriptional gene silencing of endogenous coding genes by using RNA surveillance mechanisms as a safeguard to protect their transcriptome profiles. The tug of war between RNA surveillance and RNA silencing ensures the appropriate partitioning of endogenous RNA substrates among these degradation pathways. Here we review recent advances on RNA quality control and its role in the suppression of RNA silencing at endogenous genes and discuss the mechanisms underlying the crosstalk among these pathways.

  14. Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi.

    PubMed

    Sijen, Titia; Plasterk, Ronald H A

    2003-11-20

    Transposable elements are stretches of DNA that can move and multiply within the genome of an organism. The Caenorhabditis elegans genome contains multiple Tc1 transposons that jump in somatic cells, but are silenced in the germ line. Many mutants that have lost this silencing have also lost the ability to execute RNA interference (RNAi), a process whereby genes are suppressed by exposure to homologous double-stranded RNA (dsRNA). Here we show how RNAi causes transposon silencing in the nematode germ line. We find evidence for transposon-derived dsRNAs, in particular to the terminal inverted repeats, and show that these RNAs may derive from read-through transcription of entire transposable elements. Small interfering RNAs of Tc1 were detected. When a germline-expressed reporter gene is fused to a stretch of Tc1 sequence, this transgene is silenced in a manner dependent on functional mutator genes (mut-7, mut-16 and pk732). These results indicate that RNAi surveillance is triggered by fortuitous read-through transcription of dispersed Tc1 copies, which can form dsRNA as a result of 'snap-back' of the terminal inverted repeats. RNAi mediated by this dsRNA silences transposase gene expression.

  15. Control of heterochromatin localization and silencing by the nuclear membrane protein Lem2

    PubMed Central

    Barrales, Ramón Ramos; Forn, Marta; Georgescu, Paula Raluca; Sarkadi, Zsuzsa; Braun, Sigurd

    2016-01-01

    Transcriptionally silent chromatin localizes to the nuclear periphery, which provides a special microenvironment for gene repression. A variety of nuclear membrane proteins interact with repressed chromatin, yet the functional role of these interactions remains poorly understood. Here, we show that, in Schizosaccharomyces pombe, the nuclear membrane protein Lem2 associates with chromatin and mediates silencing and heterochromatin localization. Unexpectedly, we found that these functions can be separated and assigned to different structural domains within Lem2, excluding a simple tethering mechanism. Chromatin association and tethering of centromeres to the periphery are mediated by the N-terminal LEM (LAP2–Emerin–MAN1) domain of Lem2, whereas telomere anchoring and heterochromatin silencing require exclusively its conserved C-terminal MSC (MAN1–Src1 C-terminal) domain. Particularly, silencing by Lem2 is epistatic with the Snf2/HDAC (histone deacetylase) repressor complex SHREC at telomeres, while its necessity can be bypassed by deleting Epe1, a JmjC protein with anti-silencing activity. Furthermore, we found that loss of Lem2 reduces heterochromatin association of SHREC, which is accompanied by increased binding of Epe1. This reveals a critical function of Lem2 in coordinating these antagonistic factors at heterochromatin. The distinct silencing and localization functions mediated by Lem2 suggest that these conserved LEM-containing proteins go beyond simple tethering to play active roles in perinuclear silencing. PMID:26744419

  16. Control of heterochromatin localization and silencing by the nuclear membrane protein Lem2.

    PubMed

    Barrales, Ramón Ramos; Forn, Marta; Georgescu, Paula Raluca; Sarkadi, Zsuzsa; Braun, Sigurd

    2016-01-15

    Transcriptionally silent chromatin localizes to the nuclear periphery, which provides a special microenvironment for gene repression. A variety of nuclear membrane proteins interact with repressed chromatin, yet the functional role of these interactions remains poorly understood. Here, we show that, in Schizosaccharomyces pombe, the nuclear membrane protein Lem2 associates with chromatin and mediates silencing and heterochromatin localization. Unexpectedly, we found that these functions can be separated and assigned to different structural domains within Lem2, excluding a simple tethering mechanism. Chromatin association and tethering of centromeres to the periphery are mediated by the N-terminal LEM (LAP2-Emerin-MAN1) domain of Lem2, whereas telomere anchoring and heterochromatin silencing require exclusively its conserved C-terminal MSC (MAN1-Src1 C-terminal) domain. Particularly, silencing by Lem2 is epistatic with the Snf2/HDAC (histone deacetylase) repressor complex SHREC at telomeres, while its necessity can be bypassed by deleting Epe1, a JmjC protein with anti-silencing activity. Furthermore, we found that loss of Lem2 reduces heterochromatin association of SHREC, which is accompanied by increased binding of Epe1. This reveals a critical function of Lem2 in coordinating these antagonistic factors at heterochromatin. The distinct silencing and localization functions mediated by Lem2 suggest that these conserved LEM-containing proteins go beyond simple tethering to play active roles in perinuclear silencing.

  17. Enhancing plant growth and fiber production by silencing GA 2-oxidase.

    PubMed

    Dayan, Jonathan; Schwarzkopf, Maayan; Avni, Adi; Aloni, Roni

    2010-05-01

    Enhancing plant height and growth rates is a principal objective of the fiber, pulp, wood and biomass product industries. Many biotechnological systems have been established to advance that task with emphasis on increasing the concentration of the plant hormone gibberellin, or on its signalling. In this respect, the most studied gibberellin biosynthesis enzyme is the GA 20-oxidase which catalyses the rate limiting step of the pathway. Overexpression of the gene resulted in an excessively high activity of the gibberellin deactivating enzyme, GA 2-oxidase. Consequently, this feedback regulation limits the intended outcome. We assume that silencing GA 2-oxidase transcription would abolish this antithetical effect, thereby allowing greater gibberellin accumulation. Here, we show that silencing the gibberellin deactivating enzyme in tobacco model plants results in a dramatic improvement of their growth characteristics, compared with the wild type and GA 20-oxidase over-expressing plants. Moreover, the number of xylem fiber cells in the silenced lines exceeded that of GA 20-oxidase over-expressing plants, potentially, making GA 2-oxidase silencing more profitable for the wood and fiber industries. Interestingly, crossing GA 20-oxidase over-expressing plants with GA 2-oxidase silenced plants did not yield consequential additive effects. Our findings unveil the benefits of silencing GA 2-oxidase to substantially increase tobacco growth and fiber production, which suggest using this approach in cultivated forest plantations and industrial herbaceous plants, worldwide.

  18. ATR acts stage specifically to regulate multiple aspects of mammalian meiotic silencing.

    PubMed

    Royo, Hélène; Prosser, Haydn; Ruzankina, Yaroslava; Mahadevaiah, Shantha K; Cloutier, Jeffrey M; Baumann, Marek; Fukuda, Tomoyuki; Höög, Christer; Tóth, Attila; de Rooij, Dirk G; Bradley, Allan; Brown, Eric J; Turner, James M A

    2013-07-01

    In mammals, homologs that fail to synapse during meiosis are transcriptionally inactivated. This process, meiotic silencing, drives inactivation of the heterologous XY bivalent in male germ cells (meiotic sex chromosome inactivation [MSCI]) and is thought to act as a meiotic surveillance mechanism. The checkpoint protein ATM and Rad3-related (ATR) localizes to unsynapsed chromosomes, but its role in the initiation and maintenance of meiotic silencing is unknown. Here we show that ATR has multiple roles in silencing. ATR first regulates HORMA (Hop1, Rev7, and Mad2) domain protein HORMAD1/2 phosphorylation and localization of breast cancer I (BRCA1) and ATR cofactors ATR-interacting peptide (ATRIP)/topoisomerase 2-binding protein 1 (TOPBP1) at unsynapsed axes. Later, it acts as an adaptor, transducing signaling at unsynapsed axes into surrounding chromatin in a manner that requires interdependence with mediator of DNA damage checkpoint 1 (MDC1) and H2AFX. Finally, ATR catalyzes histone H2AFX phosphorylation, the epigenetic event leading to gene inactivation. Using a novel genetic strategy in which MSCI is used to silence a chosen gene in pachytene, we show that ATR depletion does not disrupt the maintenance of silencing and that silencing comprises two phases: The first is dynamic and reversible, and the second is stable and irreversible. Our work identifies a role for ATR in the epigenetic regulation of gene expression and presents a new technique for ablating gene function in the germline.

  19. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA

    PubMed Central

    Decker, Logan M.; Xiao, Hua; Boone, Erin C.; Vierling, Michael M.; Shanker, Benjamin S.; Kingston, Shanika L.; Boone, Shannon F.; Haynes, Jackson B.; Shiu, Patrick K.T.

    2017-01-01

    In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD), which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi) proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC). Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors. PMID:28179391

  20. Epigenetic silencing of endogenous repetitive sequences by MORPHEUS' MOLECULE1 in Arabidopsis thaliana.

    PubMed

    Habu, Yoshiki

    2010-10-01

    Morpheus' molecule1 (MOM1) is a plant-specific epigenetic regulator of transcriptional gene silencing. Mutants of MOM1 release silencing of subsets of endogenous repetitive elements and transgenes without affecting their cytosine methylation status. Although MOM1 is evolutionarily related to chromodomain helicase DNA binding protein3 (CHD3), a family of chromatin remodeling proteins involved in repression of gene expression, MOM1 does not carry the functional ATPase/helicase domain essential for chromatin remodeling activity, and therefore, its mode of action is unknown. We recently performed a genome-wide survey for endogenous targets silenced by MOM1 and identified loci that are concentrated around centromeres and rich in sequences homologous to the 24-nt small interfering RNAs (siRNAs) that accumulate in wild type plants. Further and independent analyses indicated that the degree of contribution of MOM1 to maintenance of the silent states varies in different loci and that other silencing machineries, including those in the RNA-directed DNA methylation (RdDM) pathway, interact genetically with MOM1. In this short article, I review what we know about MOM1 and discuss its possible functions in silencing through examination of other silencing factors that interact genetically with MOM1.

  1. Chromatin and Transcription in Yeast

    PubMed Central

    Rando, Oliver J.; Winston, Fred

    2012-01-01

    Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

  2. Transcriptional regulation at the yeast nuclear envelope

    PubMed Central

    Steglich, Babett; Sazer, Shelley; Ekwall, Karl

    2013-01-01

    The spatial organization of the genome inside the nucleus affects many nuclear processes, such as DNA replication, DNA repair, and gene transcription. In metazoans, the nuclear periphery harbors mainly repressed genes that associate with the nuclear lamina. This review discusses how peripheral positioning is connected to transcriptional regulation in yeasts. Tethering of reporter genes to the nuclear envelope was found to result in transcriptional silencing. Similarly, repression of the silent mating type loci and subtelomeric genes is influenced by their position close to the nuclear envelope. In contrast, active genes are bound by nucleoporins and inducible genes associate with the nuclear pore complex upon activation. Taken together, these results portray the nuclear envelope as a platform for transcriptional regulation, both through activation at nuclear pores and silencing at the nuclear envelope. PMID:24021962

  3. Silencing and trans-activation of the mouse IL-2 gene in Xenopus oocytes by proteins from resting and mitogen-induced primary T-lymphocytes.

    PubMed Central

    Mouzaki, A; Weil, R; Muster, L; Rungger, D

    1991-01-01

    The Xenopus oocyte system was used to test functionally, putative trans-active elements involved in the transcriptional control of the mouse interleukin-2 (IL-2) gene in resting and mitogen-induced primary T-lymphocytes. The IL-2 gene injected into the oocyte is active over a wide range of DNA concentrations. This basal activity is silenced by the addition of protein extracts from G0-arrested spleen cells. Extracts from 8 h-stimulated spleen cells do not silence but moderately increase transcription over basal level. When IL-2 transcription is silenced first by an injection of extract from resting spleen cells, the addition of proteins from stimulated cells results in a strong increase in transcription (derepression). Use of proteins from purified splenic T-lymphocytes shows that both silencer(s) and activator(s) are contributed by these cells. Extracts from control tissues have neither a silencing nor stimulatory effect. None of the proteins tested affects the activities of co-injected control genes. Injections with IL-2 promoter mutants indicate that the main target sequence of the silencing and activating factors is a purine region (Pu-box) lying between positions -261 and -292 upstream of the IL-2 gene. Bandshift assays show differential binding of the Pu-box with proteins from resting or activated T-cells. Images PMID:2026141

  4. Has medical education killed "silence"?

    PubMed

    Lee, Shuh Shing

    2017-04-01

    There is an ignorance of "silence" observed from student selection methods to teaching and learning approaches. While selecting the candidates with suitable values to medical schools is crucial, most methods are unable to address fairness issue toward students from some disadvantaged background or certain personality specifically introversion. Similarly, teaching and learning approaches have shifted away from didactic to a more discursive methods such as brainstorming, team-based learning and case-based learning. These methods emphasize active participation and communication with team members, but having more discussion does not indicate that deep learning has taken place. Majority of these approaches require students to complete a task within an allocated time frame. Therefore, most of the time is utilized to complete a task instead of learning how to acquire a skill or learning how to learn. Important "silent" skills such as observation, reasoning process, and listening skills, are given less time or almost none due to time constraint within these discursive approaches, although these skills are extremely important as a doctor. Hence, it is time to think about on how best to balance the use of silence and externalize thought processes in medical education.

  5. Virus-induced multiple gene silencing to study redundant metabolic pathways in plants: silencing the starch degradation pathway in Nicotiana benthamiana.

    PubMed

    George, Gavin M; Bauer, Rolene; Blennow, Andreas; Kossmann, Jens; Lloyd, James R

    2012-07-01

    Virus-induced gene silencing (VIGS) is a rapid technique that allows for specific and reproducible post-transcriptional degradation of targeted mRNA. The method has been proven efficient for suppression of expression of many single enzymes. The metabolic networks of plants, however, often contain isoenzymes and gene families that are able to compensate for a mutation and mask the development of a silencing phenotype. Here, we show the application of multiple gene VIGS repression for the study of these redundant biological pathways. Several genes in the starch degradation pathway [disproportionating enzyme 1; (DPE1), disproportionating enzyme 2 (DPE2), and GWD] were silenced. The functionally distinct DPE enzymes are present in alternate routes for sugar export to the cytoplasm and result in an increase in starch production when silenced individually. Simultaneous silencing of DPE1 and DPE2 in Nicotiana benthamiana resulted in a near complete suppression in starch and accumulation of malto-oligosaccharides. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. The 2b protein of Asparagus virus 2 functions as an RNA silencing suppressor against systemic silencing to prove functional synteny with related cucumoviruses.

    PubMed

    Shimura, Hanako; Masuta, Chikara; Yoshida, Naoto; Sueda, Kae; Suzuki, Masahiko

    2013-08-01

    Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus in the family Bromoviridae. We cloned the coat protein (CP) and the 2b protein (2b) genes of AV-2 isolates from asparagus plants from various regions and found that the sequence for CP and for 2b was highly conserved among the isolates, suggesting that AV-2 from around the world is almost identical. We then made an AV-2 infectious clone by simultaneous inoculation with in vitro transcripts of RNAs 1-3 of AV-2 and in vitro-synthesized CP, which is necessary for initial infection. Because 2b of cucumoviruses in Bromoviridae can suppress systemic silencing as well as local silencing, we analyzed whether there is functional synteny of 2b between AV-2 and cucumovirus. Using the AV-2 infectious clone, we here provided first evidence that Ilarvirus 2b functions as an RNA silencing suppressor; AV-2 2b has suppressor activity against systemic silencing but not local silencing. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Chromosomal Redistribution of Male-Biased Genes in Mammalian Evolution with Two Bursts of Gene Gain on the X Chromosome

    PubMed Central

    Zhang, Yong E.; Vibranovski, Maria D.; Landback, Patrick; Marais, Gabriel A. B.; Long, Manyuan

    2010-01-01

    Mammalian X chromosomes evolved under various mechanisms including sexual antagonism, the faster-X process, and meiotic sex chromosome inactivation (MSCI). These forces may contribute to nonrandom chromosomal distribution of sex-biased genes. In order to understand the evolution of gene content on the X chromosome and autosome under these forces, we dated human and mouse protein-coding genes and miRNA genes on the vertebrate phylogenetic tree. We found that the X chromosome recently acquired a burst of young male-biased genes, which is consistent with fixation of recessive male-beneficial alleles by sexual antagonism. For genes originating earlier, however, this pattern diminishes and finally reverses with an overrepresentation of the oldest male-biased genes on autosomes. MSCI contributes to this dynamic since it silences X-linked old genes but not X-linked young genes. This demasculinization process seems to be associated with feminization of the X chromosome with more X-linked old genes expressed in ovaries. Moreover, we detected another burst of gene originations after the split of eutherian mammals and opossum, and these genes were quickly incorporated into transcriptional networks of multiple tissues. Preexisting X-linked genes also show significantly higher protein-level evolution during this period compared to autosomal genes, suggesting positive selection accompanied the early evolution of mammalian X chromosomes. These two findings cast new light on the evolutionary history of the mammalian X chromosome in terms of gene gain, sequence, and expressional evolution. PMID:20957185

  8. Chromosomal redistribution of male-biased genes in mammalian evolution with two bursts of gene gain on the X chromosome.

    PubMed

    Zhang, Yong E; Vibranovski, Maria D; Landback, Patrick; Marais, Gabriel A B; Long, Manyuan

    2010-10-05

    Mammalian X chromosomes evolved under various mechanisms including sexual antagonism, the faster-X process, and meiotic sex chromosome inactivation (MSCI). These forces may contribute to nonrandom chromosomal distribution of sex-biased genes. In order to understand the evolution of gene content on the X chromosome and autosome under these forces, we dated human and mouse protein-coding genes and miRNA genes on the vertebrate phylogenetic tree. We found that the X chromosome recently acquired a burst of young male-biased genes, which is consistent with fixation of recessive male-beneficial alleles by sexual antagonism. For genes originating earlier, however, this pattern diminishes and finally reverses with an overrepresentation of the oldest male-biased genes on autosomes. MSCI contributes to this dynamic since it silences X-linked old genes but not X-linked young genes. This demasculinization process seems to be associated with feminization of the X chromosome with more X-linked old genes expressed in ovaries. Moreover, we detected another burst of gene originations after the split of eutherian mammals and opossum, and these genes were quickly incorporated into transcriptional networks of multiple tissues. Preexisting X-linked genes also show significantly higher protein-level evolution during this period compared to autosomal genes, suggesting positive selection accompanied the early evolution of mammalian X chromosomes. These two findings cast new light on the evolutionary history of the mammalian X chromosome in terms of gene gain, sequence, and expressional evolution.

  9. Coupling transcription and alternative splicing.

    PubMed

    Kornblihtt, Alberto R

    2007-01-01

    Alternative splicing regulation not only depends on the interaction of splicing factors with splicing enhancers and silencers in the pre-mRNA, but also on the coupling between transcription and splicing. This coupling is possible because splicing is often cotranscriptional and promoter identity and occupation may affect alternative splicing. We discuss here the different mechanisms by which transcription regulates alternative splicing. These include the recruitment of splicing factors to the transcribing polymerase and "kinetic coupling", which involves changes in the rate of transcriptional elongation that in turn affect the timing in which splice sites are presented to the splicing machinery. The recruitment mechanism may depend on the particular features of the carboxyl terminal domain of RNA polymerase II, whereas kinetic coupling seems to be linked to how changes in chromatin structure and other factors affect transcription elongation.

  10. Exon Silencing by UAGG Motifs in Response to Neuronal Excitation

    PubMed Central

    An, Ping; Grabowski, Paula J

    2007-01-01

    Alternative pre-mRNA splicing plays fundamental roles in neurons by generating functional diversity in proteins associated with the communication and connectivity of the synapse. The CI cassette of the NMDA R1 receptor is one of a variety of exons that show an increase in exon skipping in response to cell excitation, but the molecular nature of this splicing responsiveness is not yet understood. Here we investigate the molecular basis for the induced changes in splicing of the CI cassette exon in primary rat cortical cultures in response to KCl-induced depolarization using an expression assay with a tight neuron-specific readout. In this system, exon silencing in response to neuronal excitation was mediated by multiple UAGG-type silencing motifs, and transfer of the motifs to a constitutive exon conferred a similar responsiveness by gain of function. Biochemical analysis of protein binding to UAGG motifs in extracts prepared from treated and mock-treated cortical cultures showed an increase in nuclear hnRNP A1-RNA binding activity in parallel with excitation. Evidence for the role of the NMDA receptor and calcium signaling in the induced splicing response was shown by the use of specific antagonists, as well as cell-permeable inhibitors of signaling pathways. Finally, a wider role for exon-skipping responsiveness is shown to involve additional exons with UAGG-related silencing motifs, and transcripts involved in synaptic functions. These results suggest that, at the post-transcriptional level, excitable exons such as the CI cassette may be involved in strategies by which neurons mount adaptive responses to hyperstimulation. PMID:17298175

  11. MOM1 mediates DNA-methylation-independent silencing of repetitive sequences in Arabidopsis

    PubMed Central

    Vaillant, Isabelle; Schubert, Ingo; Tourmente, Sylvette; Mathieu, Olivier

    2006-01-01

    The heterochromatic regions around centromeres of animal and plant chromosomes are composed of tandem repetitive sequences, interspersed with transposons and transposon derivatives. These sequences are largely transcriptionally silent and highly methylated, and are associated with specifically modified histones. Although embedded in heterochromatin, Arabidopsis 5S ribosomal RNA genes are among the most highly transcribed genes. However, some 5S genes are silenced, and we show here that this silencing can be suppressed by a reduction in CG methylation. Importantly, we show that mutation of MORPHEUS' MOLECULE 1 (MOM1) releases 5S repeat silencing independently of chromatin properties, as illustrated by the absence of detectable alteration of DNA and histone H3 methylation patterns. MOM1 also prevents transcription of 180-bp satellite repeats and 106B dispersed repeats but not of transposons. Our results provide evidence that transcription of densely methylated and highly repetitive heterochromatic sequences is controlled by two distinct epigenetic silencing pathways, one dependent on and the other independent of DNA methylation. PMID:17082821

  12. TMV induces RNA decay pathways to modulate gene silencing and disease symptoms.

    PubMed

    Conti, Gabriela; Zavallo, Diego; Venturuzzi, Andrea L; Rodriguez, Maria C; Crespi, Martin; Asurmendi, Sebastian

    2017-01-01

    RNA decay pathways comprise a combination of RNA degradation mechanisms that are implicated in gene expression, development and defense responses in eukaryotes. These mechanisms are known as the RNA Quality Control or RQC pathways. In plants, another important RNA degradation mechanism is the post-transcriptional gene silencing (PTGS) mediated by small RNAs (siRNAs). Notably, the RQC pathway antagonizes PTGS by preventing the entry of dysfunctional mRNAs into the silencing pathway to avoid global degradation of mRNA by siRNAs. Viral transcripts must evade RNA degrading mechanisms, thus viruses encode PTGS suppressor proteins to counteract viral RNA silencing. Here, we demonstrate that tobacco plants infected with TMV and transgenic lines expressing TMV MP and CP (coat protein) proteins (which are not linked to the suppression of silencing) display increased transcriptional levels of RNA decay genes. These plants also showed accumulation of cytoplasmic RNA granules with altered structure, increased rates of RNA decay for transgenes and defective transgene PTGS amplification. Furthermore, knockdown of RRP41 or RRP43 RNA exosome components led to lower levels of TMV accumulation with milder symptoms after infection, several developmental defects and miRNA deregulation. Thus, we propose that TMV proteins induce RNA decay pathways (in particular exosome components) to impair antiviral PTGS and this defensive mechanism would constitute an additional counter-defense strategy that lead to disease symptoms. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  13. MOM1 mediates DNA-methylation-independent silencing of repetitive sequences in Arabidopsis.

    PubMed

    Vaillant, Isabelle; Schubert, Ingo; Tourmente, Sylvette; Mathieu, Olivier

    2006-12-01

    The heterochromatic regions around centromeres of animal and plant chromosomes are composed of tandem repetitive sequences, interspersed with transposons and transposon derivatives. These sequences are largely transcriptionally silent and highly methylated, and are associated with specifically modified histones. Although embedded in heterochromatin, Arabidopsis 5S ribosomal RNA genes are among the most highly transcribed genes. However, some 5S genes are silenced, and we show here that this silencing can be suppressed by a reduction in CG methylation. Importantly, we show that mutation of MORPHEUS' MOLECULE 1 (MOM1) releases 5S repeat silencing independently of chromatin properties, as illustrated by the absence of detectable alteration of DNA and histone H3 methylation patterns. MOM1 also prevents transcription of 180-bp satellite repeats and 106B dispersed repeats but not of transposons. Our results provide evidence that transcription of densely methylated and highly repetitive heterochromatic sequences is controlled by two distinct epigenetic silencing pathways, one dependent on and the other independent of DNA methylation.

  14. Inverted-repeat DNA: a new gene-silencing tool for seed lipid modification.

    PubMed

    Singh, S; Green, A; Stoutjesdijk, P; Liu, Q

    2000-12-01

    Post-transcriptional gene silencing (PTGS) has been successfully used to modify seed lipids in oilseed crops like soybean, canola and sunflower. Conventionally, PTGS has been induced by transforming the plants with either antisense or co-suppression constructs targeted against key seed lipid biosynthesis genes. A major drawback of this approach has been the recovery of only a modest proportion of silenced individuals from large populations of transgenic plants. In this report we show that inverted-repeat DNA constructs containing an intron encoding RNA with a hairpin structure can induce PTGS with very high frequency.

  15. Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction?

    PubMed

    Baylin, Stephen B; Ohm, Joyce E

    2006-02-01

    Chromatin alterations have been associated with all stages of tumour formation and progression. The best characterized are epigenetically mediated transcriptional-silencing events that are associated with increases in DNA methylation - particularly at promoter regions of genes that regulate important cell functions. Recent evidence indicates that epigenetic changes might 'addict' cancer cells to altered signal-transduction pathways during the early stages of tumour development. Dependence on these pathways for cell proliferation or survival allows them to acquire genetic mutations in the same pathways, providing the cell with selective advantages that promote tumour progression. Strategies to reverse epigenetic gene silencing might therefore be useful in cancer prevention and therapy.

  16. Communication to Enhance Silence: The Trappist Experience

    ERIC Educational Resources Information Center

    Jaksa, James A.; Stech, Ernest L.

    1978-01-01

    Investigates perceptions of the amount of interpersonal communication and attitudes towards communication frequency after the Trappist monk's rule of enforced silence and solitude was lifted in 1969. Concludes that increased interpersonal communication resulted in increased self-awareness and therefore more meaningful and effective silence. (MH)

  17. Silence amenity engineering: Past and present

    NASA Astrophysics Data System (ADS)

    Fujita, Hajime; Yokono, Yasuyuki

    1993-09-01

    Recent historical development of the noise control engineering, from mere noise reduction to silence amenity engineering, is reviewed, with social and psychological backgrounds behind it. Philosophical view points for fundamental approach to the silence amenity engineering and examples of noise source control in vibration and aerodynamic noises are described.

  18. Silence as the voice of trauma.

    PubMed

    Ritter, Maria

    2014-06-01

    Silence is a key to the unspoken world of the patient. Rather than interpreting silence as a defensive maneuver, the analyst may understand this disruption as a royal road to the patient's traumatic experiences. The author proposes to recognize traumatic silences in the analytic process and the transference as a re-experiencing of past, unpredictable traumatic affective states and memories. Silences in this context are both a repeat of a disconnecting experience as well as a manifestation of a silencing identification with the original silencer. The clinical material illustrates effects of a German mother's World War II (WWII) personal traumata and collective shame-based silence on her daughter's self and good object development. In the daughter's analysis, the patient and the analyst, who herself experienced similar WWII traumata, face the pain of trauma recovery and un-silencing. The author suggests that the deadening effect of past traumata may be reversed by an analytic process of re-membering and re-speaking for both the patient and analyst. This allows for a more transparent, subjective experience in the transference and a verbal integration of ego functions.

  19. Classroom Silence: Voices from Japanese EFL Learners

    ERIC Educational Resources Information Center

    Harumi, Seiko

    2011-01-01

    This article explores Japanese EFL learners' classroom silence in a Japanese EFL context. The existence of silence in second language learning contexts can be a source of conflict between students and teachers and even among students themselves. It can also be an obstacle to acquiring the target language. In order to tackle this problem and to…

  20. Classroom Silence: Voices from Japanese EFL Learners

    ERIC Educational Resources Information Center

    Harumi, Seiko

    2011-01-01

    This article explores Japanese EFL learners' classroom silence in a Japanese EFL context. The existence of silence in second language learning contexts can be a source of conflict between students and teachers and even among students themselves. It can also be an obstacle to acquiring the target language. In order to tackle this problem and to…

  1. yKu70/yKu80 and Rif1 Regulate Silencing Differentially at Telomeres in Candida glabrata▿ ‡

    PubMed Central

    Rosas-Hernández, Lluvia L.; Juárez-Reyes, Alejandro; Arroyo-Helguera, Omar E.; De Las Peñas, Alejandro; Pan, Shih-Jung; Cormack, Brendan P.; Castaño, Irene

    2008-01-01

    Candida glabrata, a common opportunistic fungal pathogen, adheres efficiently to mammalian epithelial cells in culture. This interaction in vitro depends mainly on the adhesin Epa1, one of a large family of cell wall proteins. Most of the EPA genes are located in subtelomeric regions, where they are transcriptionally repressed by silencing. In order to better characterize the transcriptional regulation of the EPA family, we have assessed the importance of C. glabrata orthologues of known regulators of subtelomeric silencing in Saccharomyces cerevisiae. To this end, we used a series of strains containing insertions of the reporter URA3 gene within different intergenic regions throughout four telomeres of C. glabrata. Using these reporter strains, we have assessed the roles of SIR2, SIR3, SIR4, HDF1 (yKu70), HDF2 (yKu80), and RIF1 in mediating silencing at four C. glabrata telomeres. We found that, whereas the SIR proteins are absolutely required for silencing of the reporter genes and the native subtelomeric EPA genes, the Rif1 and the Ku proteins regulate silencing at only a subset of the analyzed telomeres. We also mapped a cis element adjacent to the EPA3 locus that can silence a reporter gene when placed at a distance of 31 kb from the telomere. Our data show that silencing of the C. glabrata telomeres varies from telomere to telomere. In addition, recruitment of silencing proteins to the subtelomeres is likely, for certain telomeres, to depend both on the telomeric repeats and on particular discrete silencing elements. PMID:18836091

  2. Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

    PubMed Central

    Nawaz, Z; Tsai, M J; O'Malley, B W

    1995-01-01

    Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription. Images Fig. 1 Fig. 2 Fig. 4 PMID:8524830

  3. Oomycete pathogens encode RNA silencing suppressors.

    PubMed

    Qiao, Yongli; Liu, Lin; Xiong, Qin; Flores, Cristina; Wong, James; Shi, Jinxia; Wang, Xianbing; Liu, Xigang; Xiang, Qijun; Jiang, Shushu; Zhang, Fuchun; Wang, Yuanchao; Judelson, Howard S; Chen, Xuemei; Ma, Wenbo

    2013-03-01

    Effectors are essential virulence proteins produced by a broad range of parasites, including viruses, bacteria, fungi, oomycetes, protozoa, insects and nematodes. Upon entry into host cells, pathogen effectors manipulate specific physiological processes or signaling pathways to subvert host immunity. Most effectors, especially those of eukaryotic pathogens, remain functionally uncharacterized. Here, we show that two effectors from the oomycete plant pathogen Phytophthora sojae suppress RNA silencing in plants by inhibiting the biogenesis of small RNAs. Ectopic expression of these Phytophthora suppressors of RNA silencing enhances plant susceptibility to both a virus and Phytophthora, showing that some eukaryotic pathogens have evolved virulence proteins that target host RNA silencing processes to promote infection. These findings identify RNA silencing suppression as a common strategy used by pathogens across kingdoms to cause disease and are consistent with RNA silencing having key roles in host defense.

  4. Plant RNA silencing in viral defence.

    PubMed

    Pantaleo, Vitantonio

    2011-01-01

    RNA silencing is described in plants and insects as a defence mechanism against foreign nucleic acids, such as invading viruses. The RNA silencing-based antiviral defence involves the production of virus-derived small interfering RNAs and their association to effector proteins, which together drive the sequence specific inactivation of viruses. The entire process of antiviral defence 'borrows' several plant factors involved in other specialized RNA silencing endogenous pathways. Different viruses use variable strategies to infect different host plants, which render the antiviral RNA silencing a complex phenomenon far to be completely clarified. This chapter reports current advances in understanding the main steps of the plant's RNA-silencing response to viral invasion and discusses some of the key questions still to be answered.

  5. Interobserver Agreement on First-Stage Conversation Analytic Transcription

    ERIC Educational Resources Information Center

    Roberts, Felicia; Robinson, Jeffrey D.

    2004-01-01

    This investigation assesses interobserver agreement on conversation analytic (CA) transcription. Four professional CA transcribers spent a maximum of 3 hours transcribing 2.5 minutes of a previously unknown, naturally occurring, mundane telephone call. Researchers unitized transcripts into words, sounds, silences, inbreaths, outbreaths, and laugh…

  6. Interobserver Agreement on First-Stage Conversation Analytic Transcription

    ERIC Educational Resources Information Center

    Roberts, Felicia; Robinson, Jeffrey D.

    2004-01-01

    This investigation assesses interobserver agreement on conversation analytic (CA) transcription. Four professional CA transcribers spent a maximum of 3 hours transcribing 2.5 minutes of a previously unknown, naturally occurring, mundane telephone call. Researchers unitized transcripts into words, sounds, silences, inbreaths, outbreaths, and laugh…

  7. Phenotyping of VIGS-mediated gene silencing in rice using a vector derived from a DNA virus.

    PubMed

    Kant, Ravi; Dasgupta, Indranil

    2017-07-01

    Target genes in rice can be optimally silenced if inserted in antisense or hairpin orientation in the RTBV-derived VIGS vector and plants grown at 28 °C and 80% humidity after inoculation. Virus induced gene silencing (VIGS) is a method used to transiently silence genes in dicot as well as monocot plants. For the important monocot species rice, the Rice tungro bacilliform virus (RTBV)-derived VIGS system (RTBV-VIGS), which uses agroinoculation to initiate silencing, has not been standardized for optimal use. Here, using RTBV-VIGS, three sets of conditions were tested to achieve optimal silencing of the rice marker gene phytoene desaturase (pds). The effect of orientation of the insert in the RTBV-VIGS plasmid (sense, antisense and hairpin) on the silencing of the target gene was then evaluated using rice magnesium chelatase subunit H (chlH). Finally, the rice Xa21 gene, conferring resistance against bacterial leaf blight disease (BLB) was silenced using RTBV-VIGS system. In each case, real-time PCR-based assessment indicated approximately 40-80% fall in the accumulation levels of the transcripts of pds, chlH and Xa21. In the case of pds, the appearance of white streaks in the emerging leaves, and for chlH, chlorophyll levels and F v/F m ratio were assessed as phenotypes for silencing. For Xa21, the resistance levels to BLB were assessed by measuring the lesion length and the percent diseased areas of leaves, following challenge inoculation with Xanthomonas oryzae. In each case, the RTBV-MVIGS system gave rise to a discernible phenotype indicating the silencing of the respective target gene using condition III (temperature 28 °C, humidity 80% and 1 mM MES and 20 µM acetosyringone in secondary agrobacterium culture), which revealed the robustness of this gene silencing system for rice.

  8. Neuronal activity biases axon selection for myelination in vivo

    PubMed Central

    Hines, Jacob H.; Ravanelli, Andrew M.; Schwindt, Rani; Scott, Ethan K.; Appel, Bruce

    2015-01-01

    An essential feature of vertebrate neural development is ensheathment of axons with myelin, an insulating membrane formed by oligodendrocytes. Not all axons are myelinated, but mechanisms directing myelination of specific axons are unknown. Using zebrafish we show that activity-dependent secretion stabilizes myelin sheath formation on select axons. When VAMP2-dependent exocytosis is silenced in single axons, oligodendrocytes preferentially ensheath neighboring axons. Nascent sheaths formed on silenced axons are shorter in length, but when activity of neighboring axons is also suppressed, inhibition of sheath growth is relieved. Using in vivo time-lapse microscopy, we show that only 25% of oligodendrocyte processes that initiate axon wrapping are stabilized during normal development, and that initiation does not require activity. Instead, oligodendrocyte processes wrapping silenced axons are retracted more frequently. We propose that axon selection for myelination results from excessive and indiscriminate initiation of wrapping followed by refinement that is biased by activity-dependent secretion from axons. PMID:25849987

  9. A Novel Epigenetic Silencing Pathway Involving the Highly Conserved 5’-3’ Exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe

    PubMed Central

    Tucker, James Franklin; Ohle, Corina; Schermann, Géza; Bendrin, Katja; Zhang, Wei; Fischer, Tamás; Zhang, Ke

    2016-01-01

    Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3’-5’ exoribonuclease complex, the exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5’-3’ exoribonuclease Dhp1/Rat1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation in either of the two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not result in silencing defects at the main heterochromatic regions. We demonstrate that Dhp1 interacts with heterochromatic factors and is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analyses suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its RNA processing activity. The results describe the unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing and elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome. PMID:26889830

  10. Rapid reversal of translational silencing: Emerging role of microRNA degradation pathways in neuronal plasticity.

    PubMed

    Fu, Xiuping; Shah, Aparna; Baraban, Jay M

    2016-09-01

    As microRNAs silence translation, rapid reversal of this process has emerged as an attractive mechanism for driving de novo protein synthesis mediating neuronal plasticity. Herein, we summarize recent studies identifying neuronal stimuli that trigger rapid decreases in microRNA levels and reverse translational silencing of plasticity transcripts. Although these findings indicate that neuronal stimulation elicits rapid degradation of selected microRNAs, we are only beginning to decipher the molecular pathways involved. Accordingly, we present an overview of several molecular pathways implicated in mediating microRNA degradation: Lin-28, translin/trax, and MCPIP1. As these degradation pathways target distinct subsets of microRNAs, they enable neurons to reverse silencing rapidly, yet selectively. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Posttranscriptional gene silencing of gn1 in tobacco triggers accumulation of truncated gn1-derived RNA species.

    PubMed Central

    Litière, K; van Eldik, G J; Jacobs, J J; Van Montagu, M; Cornelissen, M

    1999-01-01

    Posttranscriptional silencing of basic beta-1,3-glucanase genes in the tobacco line T17 is manifested by reduced transcript levels of the gn1 transgene and homologous, endogenous basic beta-1,3-glucanase genes. An RNA ligation-mediated rapid amplification of cDNA ends (RLM-RACE) technique was used to compare the 3' termini of gn1 RNAs present in expressing (hemizygous and young homozygous) and silenced (mature homozygous) T17 plants. Full-length, polyadenylated gn1 transcripts primarily accumulated in expressing plants, whereas in silenced T17 plants, mainly 3'-truncated, nonpolyadenylated gn1 RNAs were detected. The relative abundance of these 3'-truncated gn1 RNA species gradually increased during the establishment of silencing in homozygous T17 plants. Similar 3'-truncated, nonpolyadenylated gn1 RNA products were observed in an independent case of beta-1,3-glucanase posttranscriptional gene silencing. This suggests that these 3'-truncated gn1 RNAs are a general feature of tobacco plants showing posttranscriptional silencing of the gn1 transgene. PMID:10573127

  12. Suppression of RNA Silencing by a Geminivirus Nuclear Protein, AC2, Correlates with Transactivation of Host Genes†

    PubMed Central

    Trinks, Daniela; Rajeswaran, R.; Shivaprasad, P. V.; Akbergenov, Rashid; Oakeley, Edward J.; Veluthambi, K.; Hohn, Thomas; Pooggin, Mikhail M.

    2005-01-01

    Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing. PMID:15681452

  13. Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes.

    PubMed

    Trinks, Daniela; Rajeswaran, R; Shivaprasad, P V; Akbergenov, Rashid; Oakeley, Edward J; Veluthambi, K; Hohn, Thomas; Pooggin, Mikhail M

    2005-02-01

    Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.

  14. Reactivation of developmentally silenced globin genes by forced chromatin looping

    PubMed Central

    Krivega, Ivan; Breda, Laura; Motta, Irene; Jahn, Kristen S.; Reik, Andreas; Gregory, Philip D.; Rivella, Stefano; Dean, Ann; Blobel, Gerd A.

    2014-01-01

    Summary Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggered its transcriptional reactivation. This activity depended on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting SA to the fetal γ-globin promoter in primary adult human erythroblasts increased γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications. PMID:25126789

  15. Development and application of an efficient virus-induced gene silencing system in Nicotiana tabacum using geminivirus alphasatellite*

    PubMed Central

    Huang, Chang-jun; Zhang, Tong; Li, Fang-fang; Zhang, Xin-yue; Zhou, Xue-ping

    2011-01-01

    Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assays. Here we report that the 2mDNA1, a geminivirus satellite vector, can induce efficient gene silencing in Nicotiana tabacum with Tobacco curly shoot virus. We have successfully silenced the β-glucuronidase (GUS) gene in GUS transgenic N. tabacum plants and the sulphur desaturase (Su) gene in five different N. tabacum cultivars. These pronounced and severe silencing phenotypes are persistent and ubiquitous. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants and silencing could be induced in a variety of tissues and organs including leaf, shoot, stem, root, and flower, and achieved at any growth stage. This system works well between 18–32 °C. We also silenced the NtEDS1 gene and demonstrated that NtEDS1 is essential for N gene mediated resistance against Tobacco mosaic virus in N. tabacum. The above results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in N. tabacum. PMID:21265040

  16. The gifts of silence and solitude.

    PubMed

    Schmidt Bunkers, Sandra

    2008-01-01

    In this column the author describes the importance of finding silence and solitude amid the noise and technology present today in the teaching-learning academy. Three gifts of silence and solitude are identified: the gift of comforting aloneness, the gift of vision for new horizons, and the gift of a sense of freedom. A humanbecoming perspective is used to explore the implications of these gifts. This column introduces a column by Diana Vander Woude describing her teaching-learning experience in leadership focusing on silence and solitude.

  17. Splicing Factor Spf30 Assists Exosome-Mediated Gene Silencing in Fission Yeast▿

    PubMed Central

    Bernard, Pascal; Drogat, Julie; Dheur, Sonia; Genier, Sylvie; Javerzat, Jean-Paul

    2010-01-01

    Heterochromatin assembly in fission yeast relies on the processing of cognate noncoding RNAs by both the RNA interference and the exosome degradation pathways. Recent evidence indicates that splicing factors facilitate the cotranscriptional processing of centromeric transcripts into small interfering RNAs (siRNAs). In contrast, how the exosome contributes to heterochromatin assembly and whether it also relies upon splicing factors were unknown. We provide here evidence that fission yeast Spf30 is a splicing factor involved in the exosome pathway of heterochromatin silencing. Spf30 and Dis3, the main exosome RNase, colocalize at centromeric heterochromatin and euchromatic genes. At the centromeres, Dis3 helps recruiting Spf30, whose deficiency phenocopies the dis3-54 mutant: heterochromatin is impaired, as evidenced by reduced silencing and the accumulation of polyadenylated centromeric transcripts, but the production of siRNAs appears to be unaffected. Consistent with a direct role, Spf30 binds centromeric transcripts and locates at the centromeres in an RNA-dependent manner. We propose that Spf30, bound to nascent centromeric transcripts, perhaps with other splicing factors, assists their processing by the exosome. Splicing factor intercession may thus be a common feature of gene silencing pathways. PMID:20028739

  18. Flexible tools for gene expression and silencing in tomato.

    PubMed

    Fernandez, Ana I; Viron, Nicolas; Alhagdow, Moftah; Karimi, Mansour; Jones, Matthew; Amsellem, Ziva; Sicard, Adrien; Czerednik, Anna; Angenent, Gerco; Grierson, Donald; May, Sean; Seymour, Graham; Eshed, Yuval; Lemaire-Chamley, Martine; Rothan, Christophe; Hilson, Pierre

    2009-12-01

    As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources.

  19. Patterning of Virus-Infected Glycine max Seed Coat Is Associated with Suppression of Endogenous Silencing of Chalcone Synthase Genes

    PubMed Central

    Senda, Mineo; Masuta, Chikara; Ohnishi, Shizen; Goto, Kazunori; Kasai, Atsushi; Sano, Teruo; Hong, Jin-Sung; MacFarlane, Stuart

    2004-01-01

    Most commercial Glycine max (soybean) varieties have yellow seeds because of loss of pigmentation in the seed coat. It has been suggested that inhibition of seed coat pigmentation in yellow G. max may be controlled by homology-dependent silencing of chalcone synthase (CHS) genes. Our analysis of CHS mRNA and short-interfering RNAs provide clear evidence that the inhibition of seed coat pigmentation in yellow G. max results from posttranscriptional rather than transcriptional silencing of the CHS genes. Furthermore, we show that mottling symptoms present on the seed coat of G. max plants infected with some viruses can be caused by suppression of CHS posttranscriptional gene silencing (PTGS) by a viral silencing suppressor protein. These results demonstrate that naturally occurring PTGS plays a key role in expression of a distinctive phenotype in plants and present a simple clear example of the elucidation of the molecular mechanism for viral symptom induction. PMID:15037735

  20. Patterning of virus-infected Glycine max seed coat is associated with suppression of endogenous silencing of chalcone synthase genes.

    PubMed

    Senda, Mineo; Masuta, Chikara; Ohnishi, Shizen; Goto, Kazunori; Kasai, Atsushi; Sano, Teruo; Hong, Jin-Sung; MacFarlane, Stuart

    2004-04-01

    Most commercial Glycine max (soybean) varieties have yellow seeds because of loss of pigmentation in the seed coat. It has been suggested that inhibition of seed coat pigmentation in yellow G. max may be controlled by homology-dependent silencing of chalcone synthase (CHS) genes. Our analysis of CHS mRNA and short-interfering RNAs provide clear evidence that the inhibition of seed coat pigmentation in yellow G. max results from posttranscriptional rather than transcriptional silencing of the CHS genes. Furthermore, we show that mottling symptoms present on the seed coat of G. max plants infected with some viruses can be caused by suppression of CHS posttranscriptional gene silencing (PTGS) by a viral silencing suppressor protein. These results demonstrate that naturally occurring PTGS plays a key role in expression of a distinctive phenotype in plants and present a simple clear example of the elucidation of the molecular mechanism for viral symptom induction.

  1. Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria.

    PubMed

    Zhang, Qingfeng; Siegel, T Nicolai; Martins, Rafael M; Wang, Fei; Cao, Jun; Gao, Qi; Cheng, Xiu; Jiang, Lubin; Hon, Chung-Chau; Scheidig-Benatar, Christine; Sakamoto, Hiroshi; Turner, Louise; Jensen, Anja T R; Claes, Aurelie; Guizetti, Julien; Malmquist, Nicholas A; Scherf, Artur

    2014-09-18

    Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of PfRNase II and upsA-type var genes in parasites from severe malaria patients, implying a crucial role of PfRNase II in severe malaria. Our results uncover a previously unknown type of post-transcriptional gene silencing mechanism in malaria parasites with repercussions for other organisms. Additionally, the identification of RNase II as a parasite protein controlling the expression of virulence genes involved in pathogenesis in patients with severe malaria may provide new strategies for reducing malaria mortality.

  2. Diversification of the Core RNA Interference Machinery in Chlamydomonas reinhardtii and the Role of DCL1 in Transposon Silencing

    PubMed Central

    Casas-Mollano, J. Armando; Rohr, Jennifer; Kim, Eun-Jeong; Balassa, Eniko; van Dijk, Karin; Cerutti, Heriberto

    2008-01-01

    Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms. In multicellular eukaryotes, the core components of RNA-mediated silencing have significantly expanded and diversified, resulting in partly distinct pathways for the epigenetic control of gene expression and genomic parasites. In contrast, many unicellular organisms with small nuclear genomes seem to have lost entirely the RNA-silencing machinery or have retained only a basic set of components. We report here that Chlamydomonas reinhardtii, a unicellular eukaryote with a relatively large nuclear genome, has undergone extensive duplication of Dicer and Argonaute polypeptides after the divergence of the green algae and land plant lineages. Chlamydomonas encodes three Dicers and three Argonautes with DICER-LIKE1 (DCL1) and ARGONAUTE1 being more divergent than the other paralogs. Interestingly, DCL1 is uniquely involved in the post-transcriptional silencing of retrotransposons such as TOC1. Moreover, on the basis of the subcellular distribution of TOC1 small RNAs and target transcripts, this pathway most likely operates in the nucleus. However, Chlamydomonas also relies on a DCL1-independent, transcriptional silencing mechanism(s) for the maintenance of transposon repression. Our results suggest that multiple, partly redundant epigenetic processes are involved in preventing transposon mobilization in this green alga. PMID:18493041

  3. Sex-specific silencing of X-linked genes by Xist RNA.

    PubMed

    Gayen, Srimonta; Maclary, Emily; Hinten, Michael; Kalantry, Sundeep

    2016-01-19

    X-inactive specific transcript (Xist) long noncoding RNA (lncRNA) is thought to catalyze silencing of X-linked genes in cis during X-chromosome inactivation, which equalizes X-linked gene dosage between male and female mammals. To test the impact of Xist RNA on X-linked gene silencing, we ectopically induced endogenous Xist by ablating the antisense repressor Tsix in mice. We find that ectopic Xist RNA induction and subsequent X-linked gene silencing is sex specific in embryos and in differentiating embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). A higher frequency of X(ΔTsix)Y male cells displayed ectopic Xist RNA coating compared with X(ΔTsix)X female cells. This increase reflected the inability of X(ΔTsix)Y cells to efficiently silence X-linked genes compared with X(ΔTsix)X cells, despite equivalent Xist RNA induction and coating. Silencing of genes on both Xs resulted in significantly reduced proliferation and increased cell death in X(ΔTsix)X female cells relative to X(ΔTsix)Y male cells. Thus, whereas Xist RNA can inactivate the X chromosome in females it may not do so in males. We further found comparable silencing in differentiating X(ΔTsix)Y and 39,X(ΔTsix) (X(ΔTsix)O) ESCs, excluding the Y chromosome and instead implicating the X-chromosome dose as the source of the sex-specific differences. Because X(ΔTsix)X female embryonic epiblast cells and EpiSCs harbor an inactivated X chromosome prior to ectopic inactivation of the active X(ΔTsix) X chromosome, we propose that the increased expression of one or more X-inactivation escapees activates Xist and, separately, helps trigger X-linked gene silencing.

  4. Sex-specific silencing of X-linked genes by Xist RNA

    PubMed Central

    Gayen, Srimonta; Maclary, Emily; Hinten, Michael; Kalantry, Sundeep

    2016-01-01

    X-inactive specific transcript (Xist) long noncoding RNA (lncRNA) is thought to catalyze silencing of X-linked genes in cis during X-chromosome inactivation, which equalizes X-linked gene dosage between male and female mammals. To test the impact of Xist RNA on X-linked gene silencing, we ectopically induced endogenous Xist by ablating the antisense repressor Tsix in mice. We find that ectopic Xist RNA induction and subsequent X-linked gene silencing is sex specific in embryos and in differentiating embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). A higher frequency of XΔTsixY male cells displayed ectopic Xist RNA coating compared with XΔTsixX female cells. This increase reflected the inability of XΔTsixY cells to efficiently silence X-linked genes compared with XΔTsixX cells, despite equivalent Xist RNA induction and coating. Silencing of genes on both Xs resulted in significantly reduced proliferation and increased cell death in XΔTsixX female cells relative to XΔTsixY male cells. Thus, whereas Xist RNA can inactivate the X chromosome in females it may not do so in males. We further found comparable silencing in differentiating XΔTsixY and 39,XΔTsix (XΔTsixO) ESCs, excluding the Y chromosome and instead implicating the X-chromosome dose as the source of the sex-specific differences. Because XΔTsixX female embryonic epiblast cells and EpiSCs harbor an inactivated X chromosome prior to ectopic inactivation of the active XΔTsix X chromosome, we propose that the increased expression of one or more X-inactivation escapees activates Xist and, separately, helps trigger X-linked gene silencing. PMID:26739568

  5. Artificial trans-Acting siRNAs Confer Consistent and Effective Gene Silencing

    PubMed Central

    de la Luz Gutiérrez-Nava, Maria; Aukerman, Milo J.; Sakai, Hajime; Tingey, Scott V.; Williams, Robert W.

    2008-01-01

    Manipulating gene expression is critical to exploring gene function and a useful tool for altering commercial traits. Techniques such as hairpin-based RNA interference, virus-induced gene silencing, and artificial microRNAs take advantage of endogenous posttranscriptional gene silencing pathways to block translation of designated transcripts. Here we present a novel gene silencing method utilizing artificial trans-acting small interfering RNAs in Arabidopsis (Arabidopsis thaliana). Replacing the endogenous small interfering RNAs encoded in the TAS1c gene with sequences from the FAD2 gene silenced FAD2 activity to levels comparable to the fad2-1 null allele in nearly all transgenic events. Interestingly, exchanging the endogenous miR173 target sequence in TAS1c with an miR167 target sequence led to variable, inefficient silencing of FAD2, suggesting a specific requirement for the miR173 trigger for production of small interfering RNAs from the TAS1c locus. PMID:18441221

  6. Separation of stem cell maintenance and transposon silencing functions of Piwi protein.

    PubMed

    Klenov, Mikhail S; Sokolova, Olesya A; Yakushev, Evgeny Y; Stolyarenko, Anastasia D; Mikhaleva, Elena A; Lavrov, Sergey A; Gvozdev, Vladimir A

    2011-11-15

    Piwi-interacting RNAs (piRNAs) and Piwi proteins have the evolutionarily conserved function of silencing of repetitive genetic elements in germ lines. The founder of the Piwi subfamily, Drosophila nuclear Piwi protein, was also shown to be required for the maintenance of germ-line stem cells (GSCs). Hence, null mutant piwi females exhibit two types of abnormalities, overexpression of transposons and severely underdeveloped ovaries. It remained unknown whether the failure of GSC maintenance is related to transposon derepression or if GSC self-renewal and piRNA silencing are two distinct functions of the Piwi protein. We have revealed a mutation, piwi(Nt), removing the nuclear localization signal of the Piwi protein. piwi(Nt) females retain the ability of GSC self-renewal and a near-normal number of egg chambers in the ovarioles but display a drastic transposable element derepression and nuclear accumulation of their transcripts in the germ line. piwi(Nt) mutants are sterile most likely because of the disturbance of piRNA-mediated transposon silencing. Analysis of chromatin modifications in the piwi(Nt) ovaries indicated that Piwi causes chromatin silencing only of certain types of transposons, whereas others are repressed in the nuclei without their chromatin modification. Thus, Piwi nuclear localization that is required for its silencing function is not essential for the maintenance of GSCs. We suggest that the Piwi function in GSC self-renewal is independent of transposon repression and is normally realized in the cytoplasm of GSC niche cells.

  7. Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa.

    PubMed

    Catalanotto, Caterina; Pallotta, Massimiliano; ReFalo, Paul; Sachs, Matthew S; Vayssie, Laurence; Macino, Giuseppe; Cogoni, Carlo

    2004-03-01

    RNA interference (RNAi) in animals, cosuppression in plants, and quelling in fungi are homology-dependent gene silencing mechanisms in which the introduction of either double-stranded RNA (dsRNA) or transgenes induces sequence-specific mRNA degradation. These phenomena share a common genetic and mechanistic basis. The accumulation of short interfering RNA (siRNA) molecules that guide sequence-specific mRNA degradation is a common feature in both silencing mechanisms, as is the component of the RNase complex involved in mRNA cleavage. During RNAi in animal cells, dsRNA is processed into siRNA by an RNase III enzyme called Dicer. Here we show that elimination of the activity of two Dicer-like genes by mutation in the fungus Neurospora crassa eliminates transgene-induced gene silencing (quelling) and the processing of dsRNA to an siRNA form. The two Dicer-like genes appear redundant because single mutants are quelling proficient. This first demonstration of the involvement of Dicer in gene silencing induced by transgenes supports a model by which a dsRNA produced by the activity of cellular RNA-dependent RNA polymerases on transgenic transcripts is an essential intermediate of silencing.

  8. Hide and seek: how chromatin-based pathways silence retroelements in the mammalian germline

    PubMed Central

    Molaro, Antoine; Malik, Harmit S.

    2016-01-01

    Mobile elements comprise a major fraction of most mammalian genomes. To protect their fitness and stability, hosts must keep mobile elements in check in their germline. In most tissues mobile element insertions are decorated with chromatin modifications suggestive of transcriptional silencing. However, germline cells undergo massive chromatin reprogramming events, which erase repressive chromatin marks and necessitate de novo re-establishment of silencing. How do host genomes achieve the discrimination necessary for this de novo silencing? A series of recent studies have revealed aspects of the multi-pronged strategy that mammalian genomes use to identify and silence mobile elements. These strategies include the use of small RNA-guides, of specialized DNA-binding protein adaptors and of proteins that repair chromatin discontinuities caused by retroelement insertions. Genetic analyses reveal the importance of these mechanisms of protection, each of which specializes in silencing mobile elements of different evolutionary ages. Together, these strategies allow mammalian genomes to withstand the high burden of their parasites. PMID:26821364

  9. Chemical induction of hairpin RNAi molecules to silence vital genes in plant roots.

    PubMed

    Liu, Siming; Yoder, John I

    2016-11-29

    Understanding the functions encoded by plant genes can be facilitated by reducing transcript levels by hairpin RNA (hpRNA) mediated silencing. A bottleneck to this technology occurs when a gene encodes a phenotype that is necessary for cell viability and silencing the gene inhibits transformation. Here we compared the use of two chemically inducible plant promoter systems to drive hpRNA mediated gene silencing in transgenic, hairy roots. We cloned the gene encoding the Yellow Fluorescence Protein (YFP) into the dexamethasone inducible vector pOpOff2 and into the estradiol induced vector pER8. We then cloned a hpRNA targeting YFP under the regulation of the inducible promoters, transformed Medicago truncatula roots, and quantified YFP fluorescence and mRNA levels. YFP fluorescence was normal in pOpOff2 transformed roots without dexamethasone but was reduced with dexamethasone treatment. Interestingly, dexamethasone removal did not reverse YFP inhibition. YFP expression in roots transformed with pER8 was low even in the absence of inducer. We used the dexamethasone system to silence acetyl-CoA carboxylase gene and observed prolific root growth when this construct was transformed into Medicago until dexamethasone was applied. Our study shows that dexamethasone inducibility can be useful to silence vital genes in transgenic roots.

  10. Chemical induction of hairpin RNAi molecules to silence vital genes in plant roots

    PubMed Central

    Liu, Siming; Yoder, John I.

    2016-01-01

    Understanding the functions encoded by plant genes can be facilitated by reducing transcript levels by hairpin RNA (hpRNA) mediated silencing. A bottleneck to this technology occurs when a gene encodes a phenotype that is necessary for cell viability and silencing the gene inhibits transformation. Here we compared the use of two chemically inducible plant promoter systems to drive hpRNA mediated gene silencing in transgenic, hairy roots. We cloned the gene encoding the Yellow Fluorescence Protein (YFP) into the dexamethasone inducible vector pOpOff2 and into the estradiol induced vector pER8. We then cloned a hpRNA targeting YFP under the regulation of the inducible promoters, transformed Medicago truncatula roots, and quantified YFP fluorescence and mRNA levels. YFP fluorescence was normal in pOpOff2 transformed roots without dexamethasone but was reduced with dexamethasone treatment. Interestingly, dexamethasone removal did not reverse YFP inhibition. YFP expression in roots transformed with pER8 was low even in the absence of inducer. We used the dexamethasone system to silence acetyl-CoA carboxylase gene and observed prolific root growth when this construct was transformed into Medicago until dexamethasone was applied. Our study shows that dexamethasone inducibility can be useful to silence vital genes in transgenic roots. PMID:27898105

  11. Genomic analysis reveals epistatic silencing of "expensive" genes in Escherichia coli K-12.

    PubMed

    Srinivasan, Rajalakshmi; Chandraprakash, Deepti; Krishnamurthi, Revathy; Singh, Parul; Scolari, Vittore F; Krishna, Sandeep; Seshasayee, Aswin Sai Narain

    2013-08-01

    A barrier for horizontal gene transfer is high gene expression, which is metabolically expensive. Silencing of horizontally-acquired genes in the bacterium Escherichia coli is caused by the global transcriptional repressor H-NS. The activity of H-NS is enhanced or diminished by other proteins including its homologue StpA, and Hha and YdgT. The interconnections of H-NS with these regulators and their role in silencing gene expression in E. coli are not well understood on a genomic scale. In this study, we use transcriptome sequencing to show that there is a bi-layered gene silencing system - involving the homologous H-NS and StpA - operating on horizontally-acquired genes among others. We show that H-NS-repressed genes belong to two types, termed "epistatic" and "unilateral". In the absence of H-NS, the expression of "epistatically controlled genes" is repressed by StpA, whereas that of "unilaterally controlled genes" is not. Epistatic genes show a higher tendency to be non-essential and recently acquired, when compared to unilateral genes. Epistatic genes reach much higher expression levels than unilateral genes in the absence of the silencing system. Finally, epistatic genes contain more high affinity H-NS binding motifs than unilateral genes. Therefore, both the DNA binding sites of H-NS as well as the function of StpA as a backup system might be selected for silencing highly transcribable genes.

  12. Host-delivered RNAi: an effective strategy to silence genes in plant parasitic nematodes.

    PubMed

    Fairbairn, David J; Cavallaro, Antonino S; Bernard, Margaret; Mahalinga-Iyer, Janani; Graham, Michael W; Botella, José R

    2007-11-01

    Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary endoparasites that infect many plant species causing large economic losses worldwide. Available nematicides are being banned due to their toxicity or ozone-depleting properties and alternative control strategies are urgently required. We have produced transgenic tobacco (Nicotiana tabacum) plants expressing different dsRNA hairpin structures targeting a root-knot nematode (Meloidogyne javanica) putative transcription factor, MjTis11. We provide evidence that MjTis11 was consistently silenced in nematodes feeding on the roots of transgenic plants. The observed silencing was specific for MjTis11, with other sequence-unrelated genes being unaffected in the nematodes. Those transgenic plants able to induce silencing of MjTis11, also showed the presence of small interfering RNAs. Even though down-regulation of MjTis11 did not result in a lethal phenotype, this study demonstrates the feasibility of silencing root-knot nematode genes by expressing dsRNA in the host plant. Host-delivered RNA interference-triggered (HD-RNAi) silencing of parasite genes provides a novel disease resistance strategy with wide biotechnological applications. The potential of HD-RNAi is not restricted to parasitic nematodes but could be adapted to control other plant-feeding pests.

  13. Regulation of Ubx expression by epigenetic enhancer silencing in response to Ubx levels and genetic variation.

    PubMed

    Crickmore, Michael A; Ranade, Vikram; Mann, Richard S

    2009-09-01

    For gene products that must be present in cells at defined concentrations, expression levels must be tightly controlled to ensure robustness against environmental, genetic, and developmental noise. By studying the regulation of the concentration-sensitive Drosophila melanogaster Hox gene Ultrabithorax (Ubx), we found that Ubx enhancer activities respond to both increases in Ubx levels and genetic background. Large, transient increases in Ubx levels are capable of silencing all enhancer input into Ubx transcription, resulting in the complete silencing of this gene. Small increases in Ubx levels, brought about by duplications of the Ubx locus, cause sporadic silencing of subsets of Ubx enhancers. Ubx enhancer silencing can also be induced by outcrossing laboratory stocks to D. melanogaster strains established from wild flies from around the world. These results suggest that enhancer activities are not rigidly determined, but instead are sensitive to genetic background. Together, these findings suggest that enhancer silencing may be used to maintain gene product levels within the correct range in response to natural genetic variation.

  14. Transcriptome analysis of antigenic variation in Plasmodium falciparum - var silencing is not dependent on antisense RNA

    PubMed Central

    Ralph, Stuart A; Bischoff, Emmanuel; Mattei, Denise; Sismeiro, Odile; Dillies, Marie-Agnès; Guigon, Ghislaine; Coppee, Jean-Yves; David, Peter H; Scherf, Artur

    2005-01-01

    Background Plasmodium falciparum, the causative agent of the most severe form of malaria, undergoes antigenic variation through successive presentation of a family of antigens on the surface of parasitized erythrocytes. These antigens, known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, are subject to a mutually exclusive expression system, and are encoded by the multigene var family. The mechanism whereby inactive var genes are silenced is poorly understood. To investigate transcriptional features of this mechanism, we conducted a microarray analysis of parasites that were selected to express different var genes by adhesion to chondroitin sulfate A (CSA) or CD36. Results In addition to oligonucleotides for all predicted protein-coding genes, oligonucleotide probes specific to each known var gene of the FCR3 background were designed and added to the microarray, as well as tiled sense and antisense probes for a subset of var genes. In parasites selected for adhesion to CSA, one full-length var gene (var2csa) was strongly upregulated, as were sense RNA molecules emanating from the 3' end of a limited subset of other var genes. No global relationship between sense and antisense production of var genes was observed, but notably, some var genes had coincident high levels of both antisense and sense transcript. Conclusion Mutually exclusive expression of PfEMP1 proteins results from transcriptional silencing of non-expressed var genes. The distribution of steady-state sense and antisense RNA at var loci are not consistent with a silencing mechanism based on antisense silencing of inactive var genes. Silencing of var loci is also associated with altered regulation of genes distal to var loci. PMID:16277748

  15. Silencing of ATF2 inhibits growth of pancreatic cancer cells and enhances sensitivity to chemotherapy.

    PubMed

    Li, Mu; Wu, Xingda; Liu, Ning; Li, Xiaoying; Meng, Fanbin; Song, Shaowei

    2017-03-20

    Pancreatic cancer is one of the leading causes of cancer-related death worldwide. Activating transcription factor 2 (ATF2) is a multifunctional transcription factor, and is implicated in tumor progress, yet its role in pancreatic cancer remains unclear. In the present study, the level of ATF2 in pancreatic cancer tissues and the adjacent non-tumorous tissues was detected by quantitative real-time PCR and western blot. The roles of ATF2 in the proliferation, cell cycle, and apoptosis of pancreatic cancer cells were investigated through ATF2 silencing, and the effect of ATF2 shRNA on the sensitivity of pancreatic cancer cells to gemcitabine, an anti-tumor drug, was explored. The results of our study showed that the ATF2 level in the pancreatic cancer tissues was higher than that in the adjacent non-tumorous tissues. Silencing of ATF2 was found to inhibit proliferation, arrest cell cycle at G1 phase and induce apoptosis in pancreatic cancer cells. Moreover, ATF2 silencing enhanced gemcitabine-induced growth-inhibition and apoptosis-induction effects in pancreatic cancer cells. In summary, silencing of ATF2 inhibited the growth of pancreatic cancer cells and enhanced the anti-tumor effects of gemcitabine, suggesting that ATF2 plays a pro-survival role in pancreatic cancer. Our results also propose that a high level of ATF2 may serve as a potential biomarker of pancreatic cancer, and that ATF2 may become a potential target for anti-tumor therapy.

  16. RNA Quality Control as a Key to Suppressing RNA Silencing of Endogenous Genes in Plants

    PubMed Central

    Liu, Lin; Chen, Xuemei

    2016-01-01

    RNA quality control of endogenous RNAs is an integral part of eukaryotic gene expression and often relies on exonucleolytic degradation to eliminate dysfunctional transcripts. In parallel, exogenous and selected endogenous RNAs are degraded through RNA silencing, which is a genome defense mechanism used by many eukaryotes. In plants, RNA silencing is triggered by the production of double-stranded RNAs (dsRNAs) by RNA-DEPENDENT RNA POLYMERASEs (RDRs) and proceeds through small interfering (si) RNA-directed, ARGONAUTE (AGO)-mediated cleavage of homologous transcripts. Many studies revealed that plants avert inappropriate PTGS of endogenous coding genes by using RNA surveillance mechanisms as a safeguard to protect their transcriptome. The tug-of-war between RNA surveillance and RNA silencing ensures the appropriate partitioning of endogenous RNA substrates among these degradation pathways. Here we review recent advances on RNA quality control and its role in the suppression of RNA silencing at endogenous genes and discuss the mechanisms underlying the crosstalk among these pathways. PMID:27045817

  17. Silences: Irish women and abortion.

    PubMed

    Fletcher, R

    1995-01-01

    Notably absent from the public debate on abortion in Ireland have been the voices of women who have experienced induced abortion. Interviews with six acquaintances of the author who underwent abortion identified four themes underlying women's post-abortion silence. First, women fear public condemnation and personal rejection. Second, women are concerned that any emotional ambivalence they express about the abortion experience will be misconstrued as anti-abortion sentiment. Third, women worry that speaking out about their experience would be upsetting to friends and family. Fourth, women report frustration about the lack of a suitable public forum for voicing the complexities inherent in the abortion issue. The women's perception that their experience did not fit neatly with the rhetoric of either pro- or anti-abortion groups caused them to feel alienated from a political discourse that tends to depersonalize abortion. Although none of the women regretted the abortion decision, they continued to struggle with unresolved conflicts over taking responsibility for ending some form of life. A cycle has been created in which women do not feel safe to discuss their personal experiences until a more favorable political climate exists, yet the public perception of abortion is unlikely to change until more women's voices are heard. Feminist leaders are urged to address this dilemma.

  18. Titration and hysteresis in epigenetic chromatin silencing

    NASA Astrophysics Data System (ADS)

    Dayarian, Adel; Sengupta, Anirvan M.

    2013-06-01

    Epigenetic mechanisms of silencing via heritable chromatin modifications play a major role in gene regulation and cell fate specification. We consider a model of epigenetic chromatin silencing in budding yeast and study the bifurcation diagram and characterize the bistable and the monostable regimes. The main focus of this paper is to examine how the perturbations altering the activity of histone modifying enzymes affect the epigenetic states. We analyze the implications of having the total number of silencing proteins, given by the sum of proteins bound to the nucleosomes and the ones available in the ambient, to be constant. This constraint couples different regions of chromatin through the shared reservoir of ambient silencing proteins. We show that the response of the system to perturbations depends dramatically on the titration effect caused by the above constraint. In particular, for a certain range of overall abundance of silencing proteins, the hysteresis loop changes qualitatively with certain jump replaced by continuous merger of different states. In addition, we find a nonmonotonic dependence of gene expression on the rate of histone deacetylation activity of Sir2. We discuss how these qualitative predictions of our model could be compared with experimental studies of the yeast system under anti-silencing drugs.

  19. Recalibrating Academic Bias

    ERIC Educational Resources Information Center

    Yancey, George

    2012-01-01

    Whether political and/or religious academic bias exists is a question with important ramifications for the educational institutions. Those arguing for the presence of such bias contend that political conservatives and the highly religious in academia are marginalized and face discrimination. The question of academic bias tends to be cast in a…

  20. Oaths and hypothetical bias.

    PubMed

    Stevens, T H; Tabatabaei, Maryam; Lass, Daniel

    2013-09-30

    Results from experiments using an oath to eliminate hypothetical bias in stated preference valuation are presented. An oath has several potential advantages relative to other methods for reducing hypothetical bias. Our empirical results suggest that with an oath, mean hypothetical payments are not different from mean actual payments and that when controlling for experimental participants' characteristics using regression analyses, the oath eliminated hypothetical bias.

  1. Demonstrating the Correspondence Bias

    ERIC Educational Resources Information Center

    Howell, Jennifer L.; Shepperd, James A.

    2011-01-01

    Among the best-known and most robust biases in person perception is the correspondence bias--the tendency for people to make dispositional, rather than situational, attributions for an actor's behavior. The correspondence bias appears in virtually every social psychology textbook and in many introductory psychology textbooks, yet the authors'…

  2. Demonstrating the Correspondence Bias

    ERIC Educational Resources Information Center

    Howell, Jennifer L.; Shepperd, James A.

    2011-01-01

    Among the best-known and most robust biases in person perception is the correspondence bias--the tendency for people to make dispositional, rather than situational, attributions for an actor's behavior. The correspondence bias appears in virtually every social psychology textbook and in many introductory psychology textbooks, yet the authors'…

  3. Genetic unmasking of an epigenetically silenced microRNA in human cancer cells.

    PubMed

    Lujambio, Amaia; Ropero, Santiago; Ballestar, Esteban; Fraga, Mario F; Cerrato, Celia; Setién, Fernando; Casado, Sara; Suarez-Gauthier, Ana; Sanchez-Cespedes, Montserrat; Git, Anna; Gitt, Anna; Spiteri, Inmaculada; Das, Partha P; Caldas, Carlos; Miska, Eric; Esteller, Manel

    2007-02-15

    The mechanisms underlying microRNA (miRNA) disruption in human disease are poorly understood. In cancer cells, the transcriptional silencing of tumor suppressor genes by CpG island promoter hypermethylation has emerged as a common hallmark. We wondered if the same epigenetic disruption can "hit" miRNAs in transformed cells. To address this issue, we have used cancer cells genetically deficient for the DNA methyltransferase enzymes in combination with a miRNA expression profiling. We have observed that DNA hypomethylation induces a release of miRNA silencing in cancer cells. One of the main targets is miRNA-124a, which undergoes transcriptional inactivation by CpG island hypermethylation in human tumors from different cell types. Interestingly, we functionally link the epigenetic loss of miRNA-124a with the activation of cyclin D kinase 6, a bona fide oncogenic factor, and the phosphorylation of the retinoblastoma, a tumor suppressor gene.

  4. Cancer-associated TERT promoter mutations abrogate telomerase silencing

    PubMed Central

    Chiba, Kunitoshi; Johnson, Joshua Z; Vogan, Jacob M; Wagner, Tina; Boyle, John M; Hockemeyer, Dirk

    2015-01-01

    Mutations in the human telomerase reverse transcriptase (TERT) promoter a