Epigenetic differences between transgenes and endogenous genes promote transgene silencing in plants
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PROVIDER: PRJNA639789 | ENA |
REPOSITORIES: ENA
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Project description:Transgene loci that spontaneously undergo RNA silencing have been instrumental to decipher RNA silencing pathways in plants. In contrast, transgene loci that are stably expressed have been poorly characterized. Here we show that stably expressed transgene loci epigenetically differ from endogenous genes. Impairing the histone H3K4me3 demethylase JMJ14 increases DNA methylation at stably expressed transgene loci but not at endogenous genes, whereas impairing the histone H3K9me2 demethylase IBM1 increases DNA methylation at endogenous genes, but decreases DNA methylation at stably expressed transgene loci. Moreover, impairing IBM1 promotes post-transcriptional gene silencing (PTGS) at stably expressed transgene loci, whereas impairing JMJ14 prevents PTGS induced systemically by grafting onto silenced rootstocks. Given that impairing DNA methylation suppresses the effect of impairing JMJ14 and restores transgene capacity to undergo graft-induced PTGS, we propose that transgene DNA methylation prevents PTGS, likely by limiting the transcription of aberrant RNA by-products that are transformed into dsRNA by cellular RNA-dependent RNA polymerases to activate PTGS.
2021-03-25 | GSE152584 | GEO
Project description:Recent attempts to increase endogenous disease resistance of plants by overexpression of anti-fungal transgenes have shown a potential of this method. However, it has also been shown that such improvements are usually small. One of the obvious reasons for this low anti-fungal effect might be the regulation of endogenous genes in parallel. In this project, we will study the effect of anti-fungal transgenes on the endogenous gene expression. Such effects might relate to substantial equivalence which is a biosafety issue of concern to the public. The GeneChip Wheat Genome Array will be used to detect expression of defence response genes and key genes of metabolic pathways. We will use wheat plants transformed with anti-fungal gene of specific effect against a small group of seed transmitted, pathogenic fungi (KP4 against smuts and bunts). Transformed spring wheat line will be challenged by stinking smut (inhibited by KP4). The effect on the endogenous gene expression will be tested for plants grown in the field in collaboration with the USDA Department. This work will contribute to our understanding of plant defence responses in general and may allow improving strategies to strengthen these responses.
2010-06-02 | GSE22080 | GEO
Project description:The molecular basis of transgene susceptibility to silencing is poorly characterized in plants, thus we evaluated several transgene design parameters as means to reduce heritable sRNA-mediated transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposable element (TE)-like silencing of canola-biased PUFA transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three Bacillus thuringiensis (Bt) crystal proteins tested the impact of CDSs recoding using different codon bias tables. Transgenes with the higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.
2017-10-30 | GSE103037 | GEO
Project description:The molecular basis of transgene susceptibility to silencing is poorly characterized in plants, thus we evaluated several transgene design parameters as means to reduce heritable sRNA-mediated transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposable element (TE)-like silencing of canola-biased PUFA transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three Bacillus thuringiensis (Bt) crystal proteins tested the impact of CDSs recoding using different codon bias tables. Transgenes with the higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.
2017-10-30 | GSE76406 | GEO
Project description:Recent attempts to increase endogenous disease resistance of plants by overexpression of anti-fungal transgenes have shown a potential of this method. However, it has also been shown that such improvements are usually small. One of the obvious reasons for this low anti-fungal effect might be the regulation of endogenous genes in parallel. In this project, we will study the effect of anti-fungal transgenes on the endogenous gene expression. Such effects might relate to substantial equivalence which is a biosafety issue of concern to the public. The GeneChip Wheat Genome Array will be used to detect expression of defence response genes and key genes of metabolic pathways. We will use wheat plants transformed with anti-fungal gene of specific effect against a small group of seed transmitted, pathogenic fungi (KP4 against smuts and bunts). Transformed spring wheat line will be challenged by stinking smut (inhibited by KP4). The effect on the endogenous gene expression will be tested for plants grown in the field in collaboration with the USDA Department. This work will contribute to our understanding of plant defence responses in general and may allow improving strategies to strengthen these responses. Teliospores of pathogenic races T-1, T-5 and T-16 of T. caries provided by a collection in Aberdeen, ID, USA were used for the tests. Seeds of the genetically engineered Swiss spring wheat variety Greina (GrKP4) and the null-segregant control line (Gr0) were coated with spores and Individual plants were scored for bunt symptoms. For microarray analysis only samples inoculated with T1 and T16 were used.
2010-06-08 | E-GEOD-22080 | biostudies-arrayexpress
Project description: Not available
| S-EPMC8119426 | biostudies-literature
Project description:In eukaryotic cells, nucleo-cytoplasmic trafficking of macromolecules across the nuclear envelope is an essential process that ensures a rapid exchange of different cellular components, including proteins and RNAs, between the nucleus and cytoplasm. The significance of the nucleo-cytoplasmic trafficking of chromatin regulators in regulating DNA methylation and gene silencing is not well understood. Here, using a genetic screen, we identified XPO1A, one of the nuclear export receptors in Arabidopsis, as an anti-silencing factor that protects transgenes from transcriptional silencing. Loss-of-function of XPO1A leads to locus-specific DNA hypermethylation at transgene promoters and some endogenous loci. We found that XPO1A directly interacts with histone deacetylase HDA6 in vivo and the xpo1a mutation causes increased nuclear retention of HDA6 protein, resulting in reduced histone acetylation and enhanced transgene silencing. Our results revealed a new mechanism of epigenetic regulation through the modulation of XPO1A-dependent nucleo-cytoplasm partitioning of a chromatin regulator.
2019-01-12 | GSE119098 | GEO
Project description:PIWI interacting RNAs (piRNAs) provide defense against transposable element (TE) expansion in the germline of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the LINE-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions which normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germline. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences — not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.
2013-04-25 | GSE41780 | GEO
Project description:Next-generation Illumina sequencing technology was used to analyze small RNA associated with post-transcriptional gene silencing induced by intron-spliced hairpin RNA (ihpRNA) in Arabidopsis. 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 the specificity of the inhibition of gene expression. Despite its broad utility as a research tool, IR-PTGS is only a partially understood mechanism of RNA silencing in plants. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE SYNTHASE (CHS) gene fragments. The levels of PTGS were dependent on the orientation and position of the fragment in the IR construct. To investigate these differences, we characterized the sRNA profiles by Illumina sequencing of seven libraries generated from transgenic families showing different levels of IR-PTGS. Mapping of sRNA sequences to their corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation. Analyses of these patterns and peaks revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. Our observations provide new guidelines for designing constructs to increase the efficiency of IR-PTGS. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes. Sequencing of small RNA from Arabidopsis plants transformed with ihpRNA constructs. Seven small RNA libraries were sequenced: Lib 0, made from non-transgenic Arabidopsis plants, and Libs 1-6, made from plants transformed with different configurations of ihpRNA.
2012-06-26 | E-GEOD-38922 | biostudies-arrayexpress
Project description:In the nematode Caenorhabditis elegans, different small RNA-dependent gene silencing mechanisms act in the germline to initiate transgenerational gene silencing. Piwi-interacting RNAs (piRNAs) can initiate transposon and gene silencing by acting upstream of endogenous short interfering RNAs (siRNAs), which engage a nuclear RNA interference (RNAi) pathway to trigger transcriptional gene silencing. Once gene silencing has been established, it can be stably maintained over multiple generations without the requirement of the initial trigger and is also referred to as RNAe or paramutation. This heritable silencing depends on the integrity of the nuclear RNAi pathway. However, the exact mechanism by which silencing is maintained across generations is not understood.Here we demonstrate that silencing of piRNA targets involves the production of two distinct classes of small RNAs with different genetic requirements. The first class, secondary siRNAs, are localized close to the direct target site for piRNAs. Nuclear import of the secondary siRNAs by the Argonaute HRDE-1 leads to the production of a distinct class of small RNAs that map throughout the transcript, which we term tertiary siRNAs. Both classes of small RNAs are necessary for full repression of the target gene and can be maintained independently of the initial piRNA trigger. Consistently, we observed a form of paramutation associated with tertiary siRNAs. Once paramutated, a tertiary siRNA generating allele confers dominant silencing in the progeny regardless of its own transmission, suggesting germline-transmitted siRNAs are sufficient for multigenerational silencing. C. elegans strains containing transgenes silenced by piRNAs were crossed to strains with transgenes with similar sequences but without piRNA target sites, to investigate the spreading of silencing between transgenes mediated by small RNAs. Mutant backgrounds were used to investigate the genetic requirements for this process.
2015-04-01 | E-GEOD-66344 | biostudies-arrayexpress