It has been well established that trans-acting small RNAs guide promoter methylation leading to its inactivation and gene silencing at the transcriptional level (TGS). Here we addressed the question ...of the influence of the locus structure and epigenetic modifications of the target locus on its susceptibility for being paramutated by trans-acting small RNA molecules. Silencing was induced by crossing a 35S promoter silencer locus 271 with two different 35S-driven transgene loci, locus 2 containing a highly expressed single copy gene and locus 1 containing an inverted posttranscriptionally silenced (PTGS) repeat of this gene. Three generations of exposure to RNA signals from the 271 locus were required to complete silencing and methylation of the 35S promoter within locus 2. Segregating methylated locus 2 epialleles were obtained only from the third generation of hybrids, and this methylation was not correlated with silencing. Strikingly, only one generation was required for the PTGS locus 1 to acquire complete TGS and 35S promoter methylation. In this case, paramutated locus 1 epialleles bearing methylated and inactive 35S promoters segregated already from the first generation of hybrids. The results support the hypothesis that PTGS loci containing a palindrome structure and methylation in the coding region are more sensitive to paramutation by small RNAs and exhibit a strong tendency to formation of meiotically transmissible TGS epialleles. These features contrast with a non-methylated single copy transgenic locus that required several generations of contact with RNA silencing molecules to become imprinted in a stable epiallele.
The suppressor of cytokine signaling (SOCS) proteins, negative regulators of interferon (IFN)‐induced signaling pathways, is involved in IFN resistance of tumor cells. To improve the growth ...inhibitory effect of IFN‐β and IFN‐γ on a murine melanoma cell line, B16‐BL6, and a murine colon carcinoma cell line, Colon26 cells, SOCS‐1 and SOCS‐3 gene expression in tumor cells was downregulated by transfection of plasmid DNA expressing short hairpin RNA targeting one of these genes (pshSOCS‐1 and pshSOCS‐3, respectively). Transfection of pshSOCS‐1 significantly increased the antiproliferative effect of IFN‐γ on B16‐BL6 cells. However, any other combinations of plasmids and IFN had little effect on the growth of B16‐BL6 cells. In addition, transfection of pshSOCS‐1 and pshSOCS‐3 produced little improvement in the effect of IFN on Colon26 cells. To understand the mechanism underlining these findings, the level of SOCS gene expression was measured by real time polymerase chain reaction. Addition of IFN‐γ greatly increased the SOCS‐1 mRNA expression in B16‐BL6 cells. Taking into account the synergistic effect of pshSOCS‐1 and IFN‐γ on the growth of B16‐BL6 cells, these findings suggest that IFN‐γ‐induced high SOCS‐1 gene expression in B16‐BL6 cells significantly interferes with the antiproliferative effect of IFN‐γ. These results indicate that silencing SOCS gene expression can be an effective strategy to enhance the antitumor effect of IFN under conditions in which the SOCS gene expression is upregulated by IFN. (Cancer Sci 2008; 99: 1650–1655)
Vimentin exhibits a complex pattern of developmental- and tissue-specific expression and is aberrantly expressed in most metastatic tumors. The human vimentin promoter contains multiple DNA elements, ...some of which enhance gene expression and one that inhibits. A silencer element (at -319) binds the repressor ZBP-89. Further upstream (at -757) is an element, which acts positively in the presence of the silencer element and, thus, is referred to as an antisilencer (ASE). Previously, we showed that Stat1alpha binds to this element upon induction by IFN-gamma. However, substantial binding and reporter gene activity was still present in nontreated cells. Here, we have found that Stat3 binds to the ASE element in vitro. Transfection experiments in COS-1 cells with various vimentin promoter--reporter constructs show that gene activity is dependent upon the cotransfection and activation of Stat3. Moreover, activated Stat3 can overcome ZBP-89 repression. Coimmunoprecipitation studies demonstrate that Stat3 and ZBP-89 can interact and confocal microscopy detects these factors to be colocalized in the nucleus. Moreover, a correlation exists between the presence of activated Stat3 and vimentin expression in MDA-MB-231 cells, which is lacking in MCF7 cells where vimentin is not expressed. In the light of these results, we propose that the interaction of Stat3 and ZBP-89 may be crucial for overcoming the effects of the repressor ZBP-89, which suggests a novel mode for Stat3 gene activation.
The application of silencers in scenarios requiring both ventilation and sound insulation is extensive, and minimizing their size is an unremitting pursuit. This paper proposes a method to enhance ...the low-frequency sound absorption performance of silencer by adding thin plates on both sides of the resonant units to enhance its dissipation capability for sound waves. The addition of thin plates almost does not increase the size of the silencer but significantly lowers the peak frequency of the unit, achieving the goal of reducing the size of the silencer. As a demonstration, silencer was designed for target frequencies of 100 Hz, 200 Hz, 300 Hz, 400 Hz, and 500 Hz. Simulation result shows that the minimum transmission loss is not less than 16 dB, indicating excellent sound absorption performance, and is significantly better than ordinary silencers without baffles. This method provides an effective solution for reducing the size of silencers.
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. In most cases, the open-reading frame is disrupted which results in the absence of a ...functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and has recently been shown to correct the reading frame and restore dystrophin expression in vitro and in vivo. Specific exon skipping can be achieved using synthetic oligonucleotides or viral vectors encoding modified snRNAs, by masking important splicing sites.
We have recently demonstrated that enhanced exon skipping can be induced by a U7 snRNA carrying binding sites for the heterogeneous ribonucleoprotein A1. In DMD patient cells, bifunctional U7 snRNAs harboring silencer motifs induce complete skipping of exon 51 and thus restore dystrophin expression to near wild-type levels. Furthermore, we have confirmed the efficacy of these constructs in vivo in transgenic mice carrying the entire human DMD locus after intramuscular injection of AAV vectors encoding the bifunctional U7 snRNA. These new constructs are very promising for the optimization of therapeutic exon skipping for DMD, but also offer powerful and versatile tools to modulate pre-mRNA splicing in a wide range of applications. Here, we outline the design of these U7 snRNA constructs to achieve efficient exon skipping of the dystrophin gene. We also describe methods to evaluate the efficiency of such U7 snRNA constructs in vitro in DMD patient cells and in vivo in the transgenic hDMD mouse model, using lentiviral and recombinant adeno-associated viral vectors, respectively.
Gonadotropin‐releasing hormone (GnRH) I and II are hypothalamic decapeptides with pivotal roles in the development of reproductive competence and regulation of reproductive events. In this study, ...transcriptional regulation of the human GnRH II gene was investigated. By scanning mutation analysis coupled with transient promoter assays, the motif at −641/−636 (CATGCC, designated GII‐Sil) was identified as a repressor element. Mutation of this motif led to full restoration of promoter activity in TE671 medulloblastoma and JEG‐3 placenta choriocarcinoma cells. Supershift and chromatin immunoprecipitation assays showed in vitro and in vivo binding of NF‐κB subunit p65 and the retinoic acid receptors, RARα and RXRα, to the promoter sequences. Over‐expression of these protein factors indicated that p65 is a potent repressor, and the RARα/RXRα heterodimer is involved in the differential regulation of the GnRH II gene in neuronal and placental cells. This was confirmed by quantitative real‐time PCR. Treatment of cells with the RARα/RXRα ligands, all‐trans retinoic acid and 9‐cis‐retinoic acid, reduced and increased GnRH II gene expression in TE671 and JEG‐3 cells, respectively. Taken together, these data demonstrate the differential roles of NF‐κB p65 and RARα/RXRα, interacting with the same sequence in the promoter of the human GnRH II gene to influence gene expression in a cell‐specific manner.
The vibration and noise caused by pressure pulsation, referred to as fluid-borne vibration or fluid-borne noise, are some of the most detrimental problems in hydraulic systems. A Helmholtz silencer ...with multiple degrees of freedom was proposed to attenuate several harmonic frequencies generated in the hydraulic systems. The silencer consists of a cylindrical vessel with several chokes inside the vessel. The final goal of this research project is the development of a multiple-degree-of-freedom type of Helmholtz silencer that can be applied to hydraulic pump systems operated at different speeds. The aim of this report is to establish the design criteria of the silencer specifications. In particular, the effects of the diameter and length of the choke and the cylindrical volume on attenuation performance were analytically and experimentally investigated.
To ensure stable operation of transgenic systems and maintain reporter gene expression at a certain level, it is necessary to search for the conditions that protect the activity of regulatory ...elements. In this study, we have shown that the SV40 transcription terminators flanking the transgene protect enhancers and silencers of
Drosophila
and ensure their efficient functioning.
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