DRD1, a SWI/SNF-like chromatin remodeling protein, regulates a heat-activated transposon in Arabidopsis thaliana.

ONSEN is a heat-activated LTR retrotransposon in Arabidopsis thaliana. Screens to identify transcriptional regulatory factors of ONSEN revealed a SWI/SNF-like chromatin remodeling protein, DRD1, which cooperates with plant-specific RNA polymerase and is involved in RNA-directed DNA methylation. ONSEN transcript level was increased in the drd1 mutant relative to wild-type under heat stress, indicating that DRD1 plays a significant role in the silencing of activated ONSEN under the stress condition. The transcript level of HsfA2, which is directly involved in transcriptional activation of ONSEN, was not higher in the drd1 mutant than in the wild-type. Interestingly, no transgenerational transposition of ONSEN was observed in the drd1 mutant, even though DNA methylation levels were significantly reduced and expression levels were increased compared to the wild-type. These results suggest that other factors are involved in the regulation of ONSEN transposition in addition to the transcript level of ONSEN.

ONSEN shows different transposition activities in RdDM pathway mutants.

Most transposable elements (TEs) are tightly regulated by epigenetic mechanisms such as DNA methylation. RNA-directed DNA methylation (RdDM) is a major control mechanism of TE silencing in plants. We analyzed the transposition activity of a heat-responsive retrotransposon, ONSEN, in Arabidopsis thaliana. Transgenerational transposition was observed in RdDM pathway-deficient mutants upon heat stress. The transposition frequency was higher in the mutants of the upstream processes, but lower in the mutants of the downstream steps, of RdDM. The transposition frequency was not associated with the number of extrachromosomal ONSEN copies. Constitutive heterochromatin of interphase nuclei was dispersed upon heat stress. The degree of decondensation was higher in the RdDM mutants than in wild-type plants subjected to heat stress. We discuss the possible role of RdDM in the regulation of ONSEN transposition upon heat stress.

The effect of zebularine on the heat-activated retrotransposon ONSEN in Arabidopsis thaliana and Vigna angularis.

The Ty1/copia-like retrotransposon ONSEN is conserved among Brassica species, as well as in beans, including adzuki bean (Vigna angularis (Willd.) Ohwi & Ohashi), which is one of the economically important crops in Japan. ONSEN has acquired a heat-responsive element that is recognized by plant heat stress defense factors, resulting in its transcription and the production of full-length extrachromosomal DNA under conditions with elevated temperatures. DNA methylation plays an important role in regulating the activation of this transposon in plants. Therefore, chemical inhibition of DNA methyltransferases has been utilized to study the effect of DNA methylation on transposon activation. To understand the effect of DNA methylation on ONSEN activation, Arabidopsis thaliana and adzuki bean seedlings were treated with zebularine, which is known to be an effective chemical demethylation agent. The results showed that ONSEN transcription levels were upregulated in zebularine-treated plants. Extrachromosomal DNA of ONSEN also accumulated in the treated plants.

Characterization of a heat-activated retrotransposon in Vigna angularis.

In plants, several transposable elements are conserved across species. We found a homolog of ONSEN, which is a heat-activated retrotransposon originally isolated from Arabidopsis thaliana, in Vigna. The ONSEN-like elements (VaONS) were detected in all the analyzed Japanese accessions of Vigna angularis (adzuki bean) by Southern blot analysis. However, VaONS sequences were observed to be polymorphic in the different accessions. Interestingly, extrachromosomal DNA (ecDNA) was detected in some accessions of adzuki bean, indicating the conserved heat-activation of VaONS. Furthermore, we successfully induced retrotransposition of VaONS in adzuki plant regenerated through callus. Findings of our study should provide a new tool for molecular breeding of adzuki bean.

Characterization of a heat-activated retrotransposon in natural accessions of Arabidopsis thaliana.

Natural accessions are used for studying intraspecies genetic variation in the model plant Arabidopsis thaliana in order to address fundamental questions of evolution. Transposable elements are responsible for a wide range of mutations and play significant roles in shaping a genome over evolutionary time. In the present study, we aimed to characterize ONSEN, a heat-activated long terminal repeat (LTR) retrotransposon, in natural A. thaliana accessions. Southern blot analysis demonstrated that ONSEN was present in all the studied accessions, but the copy number was diverse. Olympia-1 contained a single ONSEN copy, located in the centromere of Chromosome 3. A premature stop codon in Olympia-1 ONSEN presumably abolishes integrase activity, which in turn presumably renders the retrotransposon non-functional. Hybridization of Col-0 with Olympia-1 showed that several ONSEN copies in Col-0 were activated by heat stress and maintained their transpositional activity in the progeny.

Inducible Transposition of a Heat-Activated Retrotransposon in Tissue Culture.

A transposition of a heat-activated retrotransposon named ONSEN required compromise of a small RNA-mediated epigenetic regulation that includes RNA-directed DNA methylation (RdDM) machinery after heat treatment. In the current study, we analyzed the transcriptional and transpositional activation of ONSEN to better understand the underlying molecular mechanism involved in the maintenance and/or induction of transposon activation in plant tissue culture. We found the transposition of heat-primed ONSEN during tissue culture independently of RdDM mutation. The heat activation of ONSEN transcripts was not significantly up-regulated in tissue culture compared with that in heat-stressed seedlings, indicating that the transposition of ONSEN was regulated independently of the transcript level. RdDM-related genes were up-regulated by heat stress in both tissue culture and seedlings. The level of DNA methylation of ONSEN did not show any change in tissue culture, and the amount of ONSEN-derived small RNAs was not affected by heat stress. The results indicated that the transposition of ONSEN was regulated by an alternative mechanism in addition to the RdDM-mediated epigenetic regulation in tissue culture. We applied the tissue culture-induced transposition of ONSEN to Japanese radish, an important breeding species of the family Brassicaceae. Several new insertions were detected in a regenerated plant derived from heat-stressed tissues and its self-fertilized progeny, revealing the possibility of molecular breeding without genetic modification.

A Stress-Activated Transposon in Arabidopsis Induces Transgenerational Abscisic Acid Insensitivity.

Transposable elements (TEs), or transposons, play an important role in adaptation. TE insertion can affect host gene function and provides a mechanism for rapid increases in genetic diversity, particularly because many TEs respond to environmental stress. In the current study, we show that the transposition of a heat-activated retrotransposon, ONSEN, generated a mutation in an abscisic acid (ABA) responsive gene, resulting in an ABA-insensitive phenotype in Arabidopsis, suggesting stress tolerance. Our results provide direct evidence that a transposon activated by environmental stress could alter the genome in a potentially positive manner. Furthermore, the ABA-insensitive phenotype was inherited when the transcription was disrupted by an ONSEN insertion, whereas ABA sensitivity was recovered when the effects of ONSEN were masked by IBM2. These results suggest that epigenetic mechanisms in host plants typically buffered the effect of a new insertion, but could selectively "turn on" TEs when stressed.

A small RNA mediated regulation of a stress-activated retrotransposon and the tissue specific transposition during the reproductive period in Arabidopsis.

Transposable elements (TEs) are key elements that facilitate genome evolution of the host organism. A number of studies have assessed the functions of TEs, which change gene expression in the host genome. Activation of TEs is controlled by epigenetic modifications such as DNA methylation and histone modifications. Several recent studies have reported that TEs can also be activated by biotic or abiotic stress in some plants. We focused on a Ty1/copia retrotransposon, ONSEN, that is activated by heat stress (HS) in Arabidopsis. We found that transcriptional activation of ONSEN was regulated by a small interfering RNA (siRNA)-related pathway, and the activation could also be induced by oxidative stress. Mutants deficient in siRNA biogenesis that were exposed to HS at the initial stages of vegetative growth showed transgenerational transposition. The transposition was also detected in the progeny, which originated from tissue that had differentiated after exposure to the HS. The results indicated that in some undifferentiated cells, transpositional activity could be maintained quite long after exposure to the HS.

Enhanced chilling tolerance at the booting stage in rice by transgenic overexpression of the ascorbate peroxidase gene, OsAPXa.

Low temperatures during the booting stage reduce rice yields by causing cold-induced male sterility. To determine whether antioxidant capacity affects the ability of rice plants to withstand chilling at the booting stage, we produced transgenic rice plants that overexpress OsAPXa and have increased APX activity. The effect of increased APX activity on the levels of H(2)O(2) and lipid peroxidation were determined by measuring H(2)O(2) levels and malondialdehyde (MDA) contents in spikelets during cold treatments at the booting stage. The levels of H(2)O(2) and the MDA content increased by 1.5-fold and twofold, respectively in WT plants subjected to a 12 °C treatment for 6 days. In contrast, transgenic lines showed small changes in H(2)O(2) levels and MDA content under cold stress, and H(2)O(2) levels and MDA content were significantly lower than in WT plants. APX activity showed negative correlations with levels of H(2)O(2) and MDA content, which increased during cold treatment. Cold tolerance at the booting stage in transgenic lines and WT plants was evaluated. Spikelet fertility was significantly higher in transgenic lines than in WT plants after a 12 °C treatment for 6 days. These results indicate that higher APX activity enhances H(2)O(2)-scavenging capacity and protects spikelets from lipid peroxidation, thereby increasing spikelet fertility under cold stress.

Gene transfer of noncleavable cell surface mutants of human CD154 induces the immune response and diminishes systemic inflammatory reactions.

CD154 (CD40-ligand) is a critical transmembrane molecule with potent immune-stimulatory properties that is used in clinical applications of gene therapy for leukemia and lymphoma. However, CD154 is cleaved into a soluble form, and high levels of sCD154 contribute to systemic inflammatory and cardiovascular diseases, suggesting a deleterious side effect of CD154 gene therapy. In this study, we engineered noncleavable mutants of human CD154 with point mutations to develop a potentially less toxic molecule in vivo. In contrast to wild-type CD154 (CD154-WT) subsequently released as sCD154, both mutants CD154-M3 and CD154-M4 were resistant to cleavage in tumor cells. Also, CD40-expressing leukemia B cells transfected with CD154-M3 mutant were highly effective stimulators in a mixed lymphocyte-leukemia reaction, indicating that CD154-M3 mutant did not lose biologic activity. In mice transplanted with tumors expressing CD154-WT, we found increased plasma levels of human sCD154 followed by various systemic inflammatory reactions such as glomerulonephritis and an increased number of infiltrating mononuclear cells in the liver. However, CD154-M3 mutant did not induce any systemic inflammatory effects in vivo. As such, the noncleavable mutant of CD154 is fully capable of inducing the immune response with less toxic properties and is a useful tool for CD154 immune gene therapy.

Gene transfer of the CD40-ligand to human dendritic cells induces NK-mediated antitumor effects against human carcinoma cells.

The CD40-ligand (CD40L) is a key molecule for the activation of dendritic cells (DCs), followed by the induction of DC maturation and cytokine production. Here we found that DC infected with adenovirus vector encoding human CD40L (CD40L-DC) displayed significantly higher levels of immune accessory molecules and IL-12 production than did uninfected cells, and that CD40L-DC produced much higher levels of IFN-gamma. To investigate whether CD40L-DC-derived these soluble factors could stimulate NK cells without physical cell-to-cell contact, we cocultured NK cells with CD40L-DC in transwell culture plates. NK cells showed up-regulated cytotoxic activity toward various squamous oral cell carcinoma (OSC-70, HSC-2, HSC-3), and we determined that both IL-12 and IFN-gamma contributed to the CD40L-DC-mediated NK cell activation. NK cells stimulated with CD40L-DC resulted in the induction of the cell surface expression of TRAIL, the production of IFN-gamma and intracellular accumulation of granzyme B. The cytotoxic activity of NK cells stimulated with CD40L-DC could be mostly inhibited by neutralizing antibody for TRAIL and completely abrogated by the combination of antibody and exocytosis inhibitor, indicating that this was mainly mediated by a TRAIL-TRAIL-receptor interaction and granule exocytosis. Moreover, CD40L-DC-activated NK cells could induce up-regulation of a death-receptor TRAIL-R2 (DR5) and down-regulation of a decoy receptor TRAIL-R3 (DcR1) on carcinoma cells. Overall, these results have revealed that CD40L-DC could activate an innate immune reaction by stimulating NK cells followed by carcinoma cells, supporting that administration of CD40L-DC may have potential as an anticancer therapy.

Bcl-xL gene transfer inhibits Bax translocation and prolongs cardiac cold preservation time in rats.

BACKGROUND: Apoptosis is an important cause of early graft loss after heart transplantation. Bcl-xL was reported to protect the heart against normothermic ischemia and reperfusion injury. In this study, we determined whether overexpression of Bcl-xL could inhibit tissue injury resulting from prolonged cold preservation followed by warm reperfusion of heart transplants. METHODS AND RESULTS: Lewis rat hearts were transduced with an adenovirus vector harboring Bcl-xL cDNA (AxCAhBclxL) 4 days before collection of tissue. After preservation in University of Wisconsin solution at 4 degrees C for 24 hours, the heart was either perfused with a Langendorff device ex vivo or used for heterotopic heart transplantation in vivo. Bcl-xL gene transfer significantly reduced the infarct size (23.0+/-2.6% versus 47.7+/-7.0% in saline control and 48.6+/-6.1% in vector control, P<0.01) after 2-hour reperfusion at 37 degrees C with the Langendorff device and significantly decreased creatine kinase release (0.82+/-0.27 IU, versus 1.57+/-0.33 and 1.50+/-0.37 IU in saline and vector controls, respectively; P<0.05). In heart transplantation, overexpression of Bcl-xL inhibited Bax translocation from the cytosol to the mitochondria, resulting in decreased cytochrome c release from the mitochondria; it also significantly decreased cardiac cell apoptosis and improved graft survival rate after long cold preservation, followed by warm reperfusion. CONCLUSIONS: Bcl-xL gene transfer inhibited the translocation of Bax and prolonged the cold preservation time of cardiac transplants. This may be a potential therapeutic method in clinical practice.