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Results for “silencing”

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PubMed1996

Cooperation at a distance between silencers and proto-silencers at the yeast HML locus.

Transcriptional repression at the silent yeast mating type loci is achieved through the formation of a particular nucleoprotein complex at specific cis-acting elements called silencers. This complex in turn appears to initiate the spreading of a histone binding protein complex into the surrounding chromatin, which restricts accessibility of the region to the transcription machinery. We have investigated long-range, cooperative effects between silencers by studying the repression of a reporter gene integrated at the HML locus flanked by various combinations of wild-type and mutated silencer sequences. Two silencers can cooperate over >4000 bp to repress transcription efficiently. More importantly, a single binding site for either the repressor activator protein 1 (Rap1), the autonomous replicating sequence (ARS) binding factor 1 (Abf1) or the origin recognition complex (ORC) can enhance the action of a distant silencer without acting as a silencer on its own. Functional cooperativity is demonstrated using a quantitative assay for repression, and varies with the affinity of the binding sites used. Since the repression mechanism is Sir dependent, the Rap1, ORC and/or Abf1 proteins bound to distant DNA elements may interact to create an interface of sufficiently high affinity such that Sir-containing complexes bind, nucleating the silent chromatin state.

Base Sequence↗

PubMed2000

Plant viral suppressors of post-transcriptional silencing do not suppress transcriptional silencing.

Homology-dependent gene silencing is a regulatory mechanism that limits RNA accumulation from affected loci either by suppression of transcription (transcriptional gene silencing, TGS) or by activation of a sequence-specific RNA degradation process (post-transcriptional gene silencing, PTGS). The P1/HC-Pro sequence of plant potyviruses and the 2b gene of the cucumber mosaic virus have been shown to interfere with PTGS. The ability of these viral suppressors of PTGS to interfere with TGS was tested using the 271 locus which imposes TGS on transgenes under 35S or 19S promoters and PTGS on the endogenous nitrite reductase gene (Nii). Both P1/HC-Pro and 2b reversed PTGS of Nii genes in 271-containing tobacco plants, but failed to reverse TGS of 35S-GUS transgenes in the same plant. P1/HC-Pro expression from a transgene also failed to suppress either the initiation or maintenance of TGS imposed by the NOSpro-silencing locus, H2. These results indicate that PTGS and TGS operate through unlinked pathways or that P1/HC-Pro and 2b interfere at step(s) in PTGS that are downstream of any common components in the two pathways. The data suggest a simple assay to identify post-transcriptionally silenced transgenic lines with the potential to be stably converted to high expressing lines.

Cucumovirus↗

PubMed2006

The silence RNA keeps: cis mechanisms of RNA mediated epigenetic silencing in mammals.

One of the fundamental questions of modern biology is to unravel how genes are switched on and off at the right time and in the correct tissues. It is well recognized that gene regulation depends on a dynamic balance between activating and repressing forces, and multiple mechanisms are involved in both gene silencing and activation. Work over the last decade has revealed that in some cases transcriptional silencing of specific genes is mediated by RNAs that specifically recruit repressing complexes to homologous DNA sequences. Examples of both cis and trans RNA driven transcriptional silencing have been reported. This review focuses on those examples of transcriptional gene silencing in which the RNA component seems to act uniquely in cis. Speculative models of how such cis acting transcripts may trigger transcriptional silencing are proposed. Future experimental testing of these and other mechanisms is important to gain a fuller understanding of how genes are regulated and to identify instances in which such mechanisms are defective, leading to disease. Understanding the basic molecular basis of these phenomena will provide us with invaluable tools for the future development of targeted therapies and drugs for those diseases in which they are faulty.

Animals↗

PubMed1997

Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing.

Agroinfiltration--the infiltration of Agrobacterium tumefaciens into intact plant levels--provides a rapid and simple way of screening large numbers of transgene constructs for silencing in response to a resident transgene. Transgenic Nicotiana sylvestris plants homozygous for the tobacco class I chitinase A gene CHN48 under the control of the cauliflower mosaic virus 35S RNA promoter (P35S) show a high incidence of postranscriptional gene silencing. We forced suspensions of A. tumefaciens, carrying P35S-CHN48 in a binary Tiplasmid vector, into wild-type and transgenic N, sylvestris leaves with a blunt-tipped plastic syringe. The infiltrated CHN48 transgene was expressed in leaves transformed with the vector alone, but not in CHN48-transformed leaves showing the silent phenotype. In contrast, expression of a chimeric P35S-E. coli beta-glucuronidase gene (uidA) infiltrated into leaves was not affected by the presence of the CHN48 transgene stably integrated in the host genome. These results show that extra copies of CHN48 are silenced by resident, silent copies of the same gene and confirm that CHN48 silencing is not the result of promoter interactions. The results also suggest that silencing of the additional CHN48 copies does not require their integration into chromosomes.

Agrobacterium tumefaciens↗

PubMed1998

Sir- and silencer-independent disruption of silencing in Saccharomyces by Sas10p.

A promoter fusion library of Saccharomyces cerevisiae genes was used to exploit phenotypes associated with altered protein dosage. We identified a novel gene, SAS10, by the ability of Sas10p, when overproduced, to disrupt silencing. The predicted Sas10p was 70,200 kD and strikingly rich in charged amino acids. Sas10p was exclusively nuclear in all stages of the cell cycle. Overproduction of Sas10p caused derepression of mating type genes at both HML and HMR, as well as of URA3, TRP1, and ADE2 when inserted near a telomere or at HMR or the rDNA locus. Repressed genes not associated with silenced chromatin were unaffected. Sas10p was essential for viability, and the termination point following Sas10p depletion was as large budded cells. Remarkably, Sas10p overproduction disrupted silencing even under conditions that bypassed the requirement for Sir proteins, ORC, and Rap1p in silencing. These data implied that Sas10p function was intimately connected with the structure of silenced chromatin.

Amino Acid Sequence↗

PubMed2006

Computational estimation and experimental verification of off-target silencing during posttranscriptional gene silencing in plants.

Successful application of posttranscriptional gene silencing (PTGS) for gene function study in both plants and animals depends on high target specificity and silencing efficiency. By computational analysis with genome and/or transcriptome sequences of 25 plant species, we predicted that about 50% to 70% of gene transcripts in plants have potential off-targets when used for PTGS that could obscure experimental results. We have developed a publicly available Web-based computational tool called siRNA Scan to identify potential off-targets during PTGS. Some of the potential off-targets obtained from this tool were tested by measuring the amount of off-target transcripts using quantitative reverse transcription-PCR. Up to 50% of the predicted off-target genes tested in plants were actually silenced when tested experimentally. Our results suggest that a high risk of off-target gene silencing exists during PTGS in plants. Our siRNA Scan tool is useful to design better constructs for PTGS by minimizing off-target gene silencing in both plants and animals.

Agrobacterium tumefaciens↗

PubMed2004

Viroid-induced RNA silencing of GFP-viroid fusion transgenes does not induce extensive spreading of methylation or transitive silencing.

Viroid infection is associated with the production of short interfering RNAs (siRNAs), a hallmark of post-transcriptional gene silencing (PTGS). However, viroid RNAs autonomously replicating in the nucleus have not been shown to trigger the degradation of homologous RNA in the cytoplasm. To investigate the potential of viroids for the induction of gene silencing, non-infectious fragments of potato spindle tuber viroid (PSTVd) cDNA were transcriptionally fused to the 3' end of the green fluorescent protein (GFP)-coding region. Introduction of such constructs into tobacco plants resulted in stable transgene expression. Upon PSTVd infection, transgene expression was suppressed and partial de novo methylation of the transgene was observed. PSTVd-specific siRNA was detected but none was found corresponding to the gfp gene. Methylation was restricted almost entirely to the PSTVd-specific part of the transgene. Neither a gfp transgene construct lacking viroid-specific elements was silenced nor was de novo methylation detected, when it was introduced into the genetic background of the PSTVd-infected plant lines containing silenced GFP:PSTVd transgenes. The absence of gfp-specific siRNAs and of significant methylation within the gfp-coding region demonstrated that neither silencing nor DNA methylation spread from the initiator region into adjacent 5' regions.

Base Sequence↗

PubMed1996

Developmental silencing of the embryonic zeta-globin gene: concerted action of the promoter and the 3'-flanking region combined with stage-specific silencing by the transcribed segment.

Globin gene switching is a well-described model of eucaryotic developmental control. In the case of the human alpha-globin gene cluster, migration of erythropoietic activity from the embryonic yolk sac to the fetal liver is parallaled by the zeta-globin gene silencing and enhanced expression of the alpha-globin genes. To map critical cis determinants of this switch, the human zeta-globin gene, the alpha-globin gene, and chimeric recombinants were introduced into the mouse genome. Consistent with previous studies, expression of the individual alpha- and zeta-globin transgenes was found to be developmentally appropriate. Contrary to current models, however, the alpha- and zeta-globin gene promoters were not sufficient to establish this control. Instead, full silencing of the zeta-globin gene required the combined activities of this promoter, transcribed region, and 3'-flanking sequences. Individually, the silencing activities of the zeta-globin gene promoter and 3'-flanking region were minimal but increased markedly when both regions were present. The zeta-globin transcribed region appeared to contribute to gene silencing by a mechanism specifically activated in definitive erythroblasts in the fetal liver. These data demonstrate that a complex set of controls, requiring at least three determinants and involving at least two independent mechanisms, is necessary for full developmental silencing of the human zeta-globin gene.

Animals↗

PubMed1999

Transcriptional and posttranscriptional silencing of rodent alpha1(I) collagen by a homologous transcriptionally self-silenced transgene.

Transient transfection of rodent fibroblasts with plasmids carrying a full-size pro-alpha1(I) collagen gene (pWTC1) results in rapid reduction of the endogenous transcripts by >90%, while the transgene mRNA is undetectable. Using deletion constructs, two adjacent 5' noncoding regions of the gene are identified as causing transcriptional silencing of the endogene in normal and v-fos-transformed cells but not in nontumorigenic revertants, which show partial relief from v-fos transformation-induced alpha1(I) gene suppression. The 3' end of the transgene carries an additional element(s), causing posttranscriptional silencing of the endogene in all cells including the revertant. Data indicate that the transgenes are transcriptionally self-silenced. Genome-integrated transgenes that are transcriptionally active also allow expression of the endogene, suggesting gene activation by chromosomal factors missing in pWTC1. Silencing is not regulated by antisense RNA. Silencing of the endogenous pro-alpha1(I) collagen gene is not linked to the level of transgene expression.

Animals↗

PubMed2003

Regulation of cholinergic gene expression by the neuron restrictive silencer factor/repressor element-1 silencing transcription factor.

The role of protein kinase A in regulating transcription of the cholinergic gene locus, which contains both the vesicular acetylcholine transporter gene and the choline acetyltransferase gene, was investigated in PC12 cells and a protein kinase A deficient PC12 mutant, A126.1B2 in which transcription of the locus is reduced. The site of action of protein kinase A was localized to a neuron restrictive silencer element/repressor element-1 (NRSE/RE-1) within the upstream region of the cholinergic gene locus. The neuron restrictive silencer factor/repressor element-1 silencing transcription factor (NRSF/REST), the transcription factor which binds to NRSE/RE-1, was expressed at similar levels in both PC12 and A126.1B2. Although nuclear extracts containing NRSF/REST from A126.1B2 exhibited binding to NRSE/RE-1, nuclear extracts from PC12 cells did not. The NRSF/REST isoform repressor element-1 silencing transcription factor-4 (REST4) was found to be expressed in PC12 cells, but not in the protein kinase A deficient PC12 cell line. REST4 inhibited the binding of NRSF/REST to NRSE/RE-1 as determined by gel mobility shift assays. Co-immunoprecipitation was used to demonstrate interaction between NRSF/REST and REST4. Expression of recombinant REST4 in the protein kinase A deficient PC12 cell line was sufficient to transcriptionally activate the cholinergic gene locus. Thus in PC12 cells protein kinase A promotes the production of REST4, which in turn de-represses of the cholinergic gene locus by inactivating the transcription repressor NRSF/REST.

Acetylcholine↗

PubMed2000

Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway.

Certain plant viruses encode suppressors of posttranscriptional gene silencing (PTGS), an adaptive antiviral defense response that limits virus replication and spread. The tobacco etch potyvirus protein, helper component-proteinase (HC-Pro), suppresses PTGS of silenced transgenes. The effect of HC-Pro on different steps of the silencing pathway was analyzed by using both transient Agrobacterium tumefaciens-based delivery and transgenic systems. HC-Pro inactivated PTGS in plants containing a preexisting silenced beta-glucuronidase (GUS) transgene. PTGS in this system was associated with both small RNA molecules (21-26 nt) corresponding to the 3' proximal region of the transcribed GUS sequence and cytosine methylation of specific sites near the 3' end of the GUS transgene. Introduction of HC-Pro into these plants resulted in loss of PTGS, loss of small RNAs, and partial loss of methylation. These results suggest that HC-Pro targets a PTGS maintenance (as opposed to an initiation or signaling) component at a point that affects accumulation of small RNAs and methylation of genomic DNA.

Agrobacterium tumefaciens↗

PubMed2003

Stable gene silencing in human monocytic cell lines using lentiviral-delivered small interference RNA. Silencing of the p110alpha isoform of phosphoinositide 3-kinase reveals differential regulation of adherence induced by 1alpha,25-dihydroxycholecalciferol and bacterial lipopolysaccharide.

Studying mononuclear phagocyte cell biology through genetic manipulation by non-viral transfection methods has been challenging due to the dual problems of low transfection efficiency and the difficulty in obtaining stable transfection. To overcome this problem, we developed a system for mediating RNA interference in monocytic cells. The p110alpha isoform of phosphoinositide 3-kinases (PI3Ks) was silenced using a lentiviral vector expressing short hairpin RNA (shRNA). This resulted in the generation of stable THP-1 and U-937 monocytic cell lines deficient in p110alpha. Notably, p110alpha was silenced without affecting levels of either the other class I(A) PI3K catalytic subunits p110beta and p110delta, or the p85alpha regulatory subunit. The role of p110alpha in mediating cell adherence was examined. Monocyte adherence induced in response to either lipopolysaccharide (LPS) or 1alpha,25-dihydroxycholecalciferol (D(3)) was blocked by the PI3K inhibitor LY294002. However, although adherence induced in response to D(3) was sensitive to silencing of p110alpha, LPS-induced adherence was not. Expression of the monocyte differentiation marker CD11b was also induced by D(3) in a PI3K-dependent manner and gene silencing using shRNA showed that p110alpha was also required for this effect. Taken together, these findings demonstrate that LPS and D(3) use distinct isoforms of class I(A) PI3K to induce functional responses and that lentiviral-mediated delivery of shRNA is a powerful approach to study monocyte biology.

Calcitriol↗

PubMed2004

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

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.

Acetylation↗

PubMed1996

Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression.

Transcriptional repression at the silent mating-type loci in yeast requires the targeting of silent information regulator (Sir) proteins through specific interactions formed at cis-acting silencer elements. We show here that a reporter gene flanked by two functional silencers is not repressed when integrated at >200 kb from a telomere. Repression is restored by creation of a new telomere 13 kb from the integrated reporter or by elevated expression of SIR1, SIR3, and/or SIR4. Coupled expression represses in an additive manner, suggesting that all three factors are in limiting concentrations. When overexpressed, Sir3 and Sir4 are dispersed throughout the nucleoplasm, in contrast to wild-type cells where they are clustered in a limited number of foci together with telomeres. Efficient silencer function thus seems to require either proximity to a pool of concentrated Sir proteins, that is, proximity to telomeres, or delocalization of the silencing factors.

Base Sequence↗

PubMed2005

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing.

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.

Animals↗

PubMed2006

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

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.

Blotting, Northern↗

PubMed2004

Regulation of the cholinergic gene locus by the repressor element-1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF).

The cholinergic gene locus is comprised of two genes, the choline acetyltransferase gene and the vesicular acetylcholine transporter gene. The vesicular acetylcholine transporter gene is located within the first intron of the choline acetyltransferase gene. This arrangement permits coordinate regulation of the locus. Protein kinase A regulates expression of the cholinergic gene locus in PC12 cells. This regulation was found to be dependent on the presence of a 21-bp DNA sequence known as the repressor element- (RE- 1)/neuron-restrictive silencer element(NRSE). Repressor element-I silencing transcription factor (REST)/ neuron-restrictive silencer factor (NRSF), which binds to the RE-I/NRSE, is a zinc finger containing transcriptional repressor that blocks the expression of many neuronal RE-I/NRSE containing genes in nonneuronal cells. However, REST/NRSF expression has also been observed in neurons as well as the PC 12 cell line used in these studies. REST/NRSF truncated isoforms were expressed in neuronal cells, suggesting they also function in regulating neuronal gene expression. A study of REST4, one of the REST/NRSF isoforms, suggests that it regulates transcription of the cholinergic gene locus by blocking the repressor activity of REST/NRSF. Protein kinase A regulation of the cholinergic gene locus in PC 12 cells can thus be attributed, at least in part, to increased synthesis of REST4, which in turn derepresses the repressor activity of REST/NRSF.

Animals↗

PubMed2003

Identification of repressor element 1 in cytochrome P450 genes and their negative regulation by RE1 silencing transcription factor/neuron-restrictive silencer factor.

RE1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) mediates transcriptional repression in many neuron-specific genes by interaction with the repressor element 1/neuron-restrictive silencing element (RE1/NRSE). This element has been identified at least in 20 neuron specific genes. REST/NRSF is highly expressed in non-neuronal tissues, where it is thought to repress gene transcription. We performed a BLAST search to look for the presence of RE1/NRSE elements in the rat cytochrome P450 genes. We identified the presence of RE1/NRSE element in the cytochrome P450 genes CYP1A1, 2A2, 2E1 and 3A2. Electrophoretic mobility shift assay and supershift assays were carried out to prove functionality of these sites and detect the interaction of REST/NRSF with this sequence. Cotransfection studies in PC12 cells with a plasmid containing the RE1 element of the CYP genes, cloned upstream of the minimal type II sodium channel promoter, in the presence of REST/NRSF, showed a marked expression inhibition of the CAT reporter gene. These data suggest that the RE1 elements that exist in these four CYP genes might be a target for the REST/NRSF transcription factor and such an interaction might play a role in the negative regulation of these genes.

Animals↗