RESUMO
RNA-directed DNA methylation, one of several RNA interference-mediated pathways in the nucleus, has been documented in plants and in human cells. Despite progress in identifying the DNA methyltransferases, histone-modifying enzymes and RNA interference proteins needed for RNA-directed DNA methylation, the mechanism remains incompletely understood. We screened for mutants defective in RNA-directed DNA methylation and silencing of a transgene promoter in Arabidopsis thaliana and identified three drd complementation groups. DRD1 is a SNF2-like protein required for RNA-directed de novo methylation. We report here that DRD2 and DRD3 correspond to the second-largest subunit and largest subunit, respectively, of a fourth class of DNA-dependent RNA polymerase (polymerase IV) that is unique to plants. DRD3 is a functionally diversified homolog of NRPD1a or SDE4, identified in a separate screen for mutants defective in post-transcriptional gene silencing. The identical DNA methylation patterns observed in all three drd mutants suggest that DRD proteins cooperate to create a substrate for RNA-directed de novo methylation.
Assuntos
Arabidopsis/enzimologia , Metilação de DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Inativação Gênica , RNA de Plantas/metabolismo , Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/genética , Dados de Sequência Molecular , Mutação , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA de Plantas/genéticaRESUMO
Chromosome termini form a specialized type of heterochromatin that is important for chromosome stability. The recent discovery of telomeric RNA transcripts in yeast and vertebrates raised the question of whether RNA-based mechanisms are involved in the formation of telomeric heterochromatin. In this study, we performed detailed analysis of chromatin structure and RNA transcription at chromosome termini in Arabidopsis. Arabidopsis telomeres display features of intermediate heterochromatin that does not extensively spread to subtelomeric regions which encode transcriptionally active genes. We also found telomeric repeat-containing transcripts arising from telomeres and centromeric loci, a portion of which are processed into small interfering RNAs. These telomeric siRNAs contribute to the maintenance of telomeric chromatin through promoting methylation of asymmetric cytosines in telomeric (CCCTAAA)(n) repeats. The formation of telomeric siRNAs and methylation of telomeres relies on the RNA-dependent DNA methylation pathway. The loss of telomeric DNA methylation in rdr2 mutants is accompanied by only a modest effect on histone heterochromatic marks, indicating that maintenance of telomeric heterochromatin in Arabidopsis is reinforced by several independent mechanisms. In conclusion, this study provides evidence for an siRNA-directed mechanism of chromatin maintenance at telomeres in Arabidopsis.
Assuntos
Arabidopsis/genética , Metilação de DNA , RNA Interferente Pequeno/genética , Telômero , Cromatina/genética , Cromossomos de Plantas , Regulação da Expressão Gênica de Plantas , Mutação , Transcrição GênicaRESUMO
Interest in transgenerational epigenetic inheritance has intensified with the boosting of knowledge on epigenetic mechanisms regulating gene expression during development and in response to internal and external signals such as biotic and abiotic stresses. Starting with an historical background of scantily documented anecdotes and their consequences, we recapitulate the information gathered during the last 60 years on naturally occurring and induced epialleles and paramutations in plants. We present the major players of epigenetic regulation and their importance in controlling stress responses. The effect of diverse stressors on the epigenetic status and its transgenerational inheritance is summarized from a mechanistic viewpoint. The consequences of transgenerational epigenetic inheritance are presented, focusing on the knowledge about its stability, and in relation to genetically fixed mutations, recombination, and genomic rearrangement. We conclude with an outlook on the importance of transgenerational inheritance for adaptation to changing environments and for practical applications. This article is part of a Special Issue entitled "Epigenetic control of cellular and developmental processes in plants".
Assuntos
Epigênese Genética , Plantas/genética , Aclimatação/genética , Adaptação Fisiológica/genética , Metilação de DNA , Epigenômica/história , Genoma de Planta , Histonas/genética , Histonas/metabolismo , História do Século XX , História do Século XXI , Mutação , Desenvolvimento Vegetal , Plantas/metabolismo , Estresse FisiológicoRESUMO
In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter, as well as the DMTase MET1 and the putative histone deacetylase HDA6 in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily. In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 and ATRX, which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Proteínas Nucleares , RNA/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Arabidopsis , Northern Blotting , Southern Blotting , Cruzamentos Genéticos , Primers do DNA , Proteínas de Ligação a DNA/genética , Metanossulfonato de Etila , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Dados de Sequência Molecular , Mutagênese , Regiões Promotoras Genéticas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA , Sulfitos , Fatores de Transcrição/genética , Transgenes/genéticaRESUMO
RNA interference is a conserved process in which double-stranded RNA is processed into 21-25 nucleotide siRNAs that trigger posttranscriptional gene silencing. In addition, plants display a phenomenon termed RNA-directed DNA methylation (RdDM) in which DNA with sequence identity to silenced RNA is de novo methylated at its cytosine residues. This methylation is not only at canonical CpG sites but also at cytosines in CpNpG and asymmetric sequence contexts. In this report, we study the role of the DRM and CMT3 DNA methyltransferase genes in the initiation and maintenance of RdDM. Neither drm nor cmt3 mutants affected the maintenance of preestablished RNA-directed CpG methylation. However, drm mutants showed a nearly complete loss of asymmetric methylation and a partial loss of CpNpG methylation. The remaining asymmetric and CpNpG methylation was dependent on the activity of CMT3, showing that DRM and CMT3 act redundantly to maintain non-CpG methylation. These DNA methyltransferases appear to act downstream of siRNAs, since drm1 drm2 cmt3 triple mutants show a lack of non-CpG methylation but elevated levels of siRNAs. Finally, we demonstrate that DRM activity is required for the initial establishment of RdDM in all sequence contexts including CpG, CpNpG, and asymmetric sites.
Assuntos
Proteínas de Arabidopsis , Ilhas de CpG/genética , Metilação de DNA , DNA-Citosina Metilases/metabolismo , Metiltransferases/metabolismo , Arabidopsis , Northern Blotting , Southern Blotting , DNA-Citosina Metilases/genética , Inativação Gênica , Metiltransferases/genética , RNA Interferente Pequeno/metabolismoRESUMO
The 'nuclear side' of RNA interference (RNAi) is increasingly recognized as an integral part of RNA-mediated gene silencing networks. Current data are consistent with the idea that epigenetic changes, such as DNA (cytosine-5) methylation and histone modifications, can be targeted to identical DNA sequences by short RNAs derived via Dicer cleavage of double-stranded RNA (dsRNA). To determine the relationships among RNA signals, DNA methylation and chromatin structure, we are carrying out a genetic analysis of RNA-mediated transcriptional gene silencing (TGS) in Arabidopsis. Results obtained so far indicate that in response to RNA signals, different site-specific DNA methyltransferases (DMTases) cooperate with each other and eventually with histone-modifying enzymes to establish and maintain a transcriptionally inactive state at a homologous target promoter. Processing of dsRNA in Arabidopsis occurs in the nucleus and in the cytoplasm, where distinct Dicer-like (DCL) activities are thought to generate functionally distinct classes of short RNAs. RNA silencing pathways thus operate throughout the cell to defend against invasive nucleic acids and to regulate genome structure and function.
Assuntos
Inativação Gênica , RNA/metabolismo , Transcrição Gênica , Animais , Metilação de DNA , Técnicas Genéticas , Heterocromatina/genética , Heterocromatina/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , RNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
Heat stress affects epigenetic gene silencing in Arabidopsis. To test for a mechanistic involvement of epigenetic regulation in heat-stress responses, we analyzed the heat tolerance of mutants defective in DNA methylation, histone modifications, chromatin-remodeling, or siRNA-based silencing pathways. Plants deficient in NRPD2, the common second-largest subunit of RNA polymerases IV and V, and in the Rpd3-type histone deacetylase HDA6 were hypersensitive to heat exposure. Microarray analysis demonstrated that NRPD2 and HDA6 have independent roles in transcriptional reprogramming in response to temperature stress. The misexpression of protein-coding genes in nrpd2 mutants recovering from heat correlated with defective epigenetic regulation of adjacent transposon remnants which involved the loss of control of heat-stress-induced read-through transcription. We provide evidence that the transcriptional response to temperature stress, at least partially, relies on the integrity of the RNA-dependent DNA methylation pathway.
Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Metilação de DNA/genética , Temperatura Alta , RNA de Plantas/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Elementos de DNA Transponíveis/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Epigênese Genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Mutação , Proteínas de Plantas/genética , Regulação para CimaAssuntos
Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , Transcrição Gênica , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/metabolismo , RNA de Cadeia Dupla/isolamento & purificação , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , RNA Interferente Pequeno/isolamento & purificaçãoRESUMO
Plants, as sessile organisms, need to sense and adapt to heterogeneous environments and have developed sophisticated responses by changing their cellular physiology, gene regulation, and genome stability. Recent work demonstrated heritable stress effects on the control of genome stability in plants--a phenomenon that was suggested to be of epigenetic nature. Here, we show that temperature and UV-B stress cause immediate and heritable changes in the epigenetic control of a silent reporter gene in Arabidopsis. This stress-mediated release of gene silencing correlated with pronounced alterations in histone occupancy and in histone H3 acetylation but did not involve adjustments in DNA methylation. We observed transmission of stress effects on reporter gene silencing to non-stressed progeny, but this effect was restricted to areas consisting of a small number of cells and limited to a few non-stressed progeny generations. Furthermore, stress-induced release of gene silencing was antagonized and reset during seed aging. The transient nature of this phenomenon highlights the ability of plants to restrict stress-induced relaxation of epigenetic control mechanisms, which likely contributes to safeguarding genome integrity.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Epigênese Genética/genética , Acetilação , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/fisiologia , Inativação Gênica/fisiologia , Histonas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The use of bacterial antibiotic resistance markers in transgenic plants raises concerns about horizontal gene transfer to soil bacteria. We report here that kanamycin resistance in Arabidopsis thaliana can be achieved by silencing an endogenous gene encoding a putative chloroplast transporter, which presumably imports kanamycin into chloroplasts to interfere with ribosomal RNA. Homologs of the transporter exist in other plant species, suggesting this strategy may be generally useful for selecting transformed plant cells.
Assuntos
Arabidopsis/genética , Farmacorresistência Bacteriana/genética , Inativação Gênica/fisiologia , Melhoramento Genético/métodos , Canamicina/administração & dosagem , Proteínas de Membrana Transportadoras/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/fisiologia , Antibacterianos/administração & dosagem , Arabidopsis/efeitos dos fármacos , Proteínas de Cloroplastos , Plantas Geneticamente Modificadas/efeitos dos fármacosRESUMO
DRD1 is a SWI/SNF-like protein that cooperates with a plant-specific RNA polymerase, Pol IVb, to facilitate RNA-directed de novo methylation and silencing of homologous DNA. Screens to identify endogenous targets of this pathway in Arabidopsis revealed intergenic regions and plant genes located primarily in euchromatin. Many putative targets are near retrotransposon LTRs or other intergenic sequences that encode short RNAs, which might epigenetically regulate adjacent genes. Consistent with this, derepression of a solo LTR in drd1 and pol IVb mutants was accompanied by reduced cytosine methylation and transcriptional upregulation of neighboring sequences. The solo LTR and several other LTRs that flank reactivated targets are associated with euchromatic histone modifications but little or no H3K9 dimethylation, a hallmark of constitutive heterochromatin. By contrast, LTRs of retrotransposons that remain silent in the mutants despite reduced cytosine methylation lack euchromatic marks and have H3K9 dimethylation. We propose that DRD1 and Pol IVb establish a basal level of silencing that can potentially be reversed in euchromatin, and further reinforced in heterochromatin by other proteins that induce more stable modifications.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Metilação de DNA , DNA Polimerase beta/metabolismo , RNA de Plantas/metabolismo , Proteínas de Arabidopsis/metabolismo , Imunoprecipitação da Cromatina , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Modelos Genéticos , Mutação/genética , Sequências Repetidas Terminais/genética , Regulação para Cima/genéticaRESUMO
DRD1 is a SNF2-like protein previously identified in a screen for mutants defective in RNA-directed DNA methylation of a seed promoter in Arabidopsis. Although the initial study established a role for DRD1 in RNA-directed DNA methylation, it did not address whether DRD1 is needed for de novo or maintenance methylation, or whether it is required for methylation of other target sequences. We show here that DRD1 is essential for RNA-directed de novo methylation and acts on different target promoters. In addition, an unanticipated role for DRD1 in erasure of CG methylation was shown when investigating maintenance methylation after segregating away the silencing trigger. DRD1 is unique among known SNF2-like proteins in facilitating not only de novo methylation of target sequences in response to RNA signals, but also loss of methylation when the silencing inducer is withdrawn. The opposing roles of DRD1 could contribute to the dynamic regulation of DNA methylation.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Ilhas de CpG , Metilação de DNA , RNA Polimerases Dirigidas por DNA/fisiologia , Aminoácido Oxirredutases/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/genética , Inativação Gênica , Regiões Promotoras Genéticas/genética , RNA de Plantas/metabolismoRESUMO
Analyses of transgene silencing phenomena in plants and other organisms have revealed the existence of epigenetic silencing mechanisms that are based on recognition of nucleic acid sequence homology at either the DNA or RNA level. Common triggers of homology-dependent gene silencing include inverted DNA repeats and double-stranded RNA, a versatile silencing molecule that can induce both degradation of homologous RNA in the cytoplasm and methylation of homologous DNA sequences in the nucleus. Inverted repeats might be frequently associated with silencing because they can potentially interact in cis and in trans to trigger DNA methylation via homologous DNA pairing, or they can be transcribed to produce double-stranded RNA. Homology-dependent gene silencing mechanisms are ideally suited for countering natural parasitic sequences such as transposable elements and viruses, which are usually present in multiple copies and/or produce double-stranded RNA during replication. These silencing mechanisms can thus be regarded as host defense strategies to foreign or invasive nucleic acids. The high content of transposable elements and, in some cases, endogenous viruses in many plant genomes suggests that host defenses do not always prevail over invasive sequences. During evolution, slightly faulty genome defense responses probably allowed transposable elements and viral sequences to accumulate gradually in host chromosomes and to invade host genes. Possible beneficial consequences of this "foreign" DNA buildup include the establishment of genome defense-derived epigenetic control mechanisms for regulating host gene expression and acquired hereditary immunity to some viruses.
Assuntos
Inativação Gênica , Plantas/genética , Animais , Evolução Biológica , Metilação de DNA , DNA de Plantas/genética , Mecanismo Genético de Compensação de Dose , Feminino , Impressão Genômica , Masculino , Mamíferos/genética , Modelos Genéticos , Vírus de Plantas/genética , Processamento Pós-Transcricional do RNA , RNA de Plantas/genética , Receptor IGF Tipo 2/genéticaRESUMO
A genetic screen for mutants defective in RNA-directed DNA methylation and transcriptional silencing of the constitutive nopaline synthase (NOS) promoter in Arabidopsis identified two independent mutations in the gene encoding the DNA methyltransferase MET1. Both mutant alleles are disrupted structurally in the MET1 catalytic domain, suggesting that they are complete loss of function alleles. Experiments designed to test the effect of a met1 mutation on both RNA-directed de novo and maintenance methylation of the target NOS promoter revealed in each case approximately wild type levels of non-CG methylation together with significant reductions of CG methylation. These results confirm a requirement for MET1 to maintain CG methylation induced by RNA. In addition, the failure to establish full CG methylation in met1 mutants, despite normal RNA-directed de novo methylation of Cs in other sequence contexts, indicates that MET1 is required for full de novo methylation of CG dinucleotides. We discuss MET1 as a site-specific DNA methyltransferase that is able to maintain CG methylation during DNA replication and contribute to CG de novo methylation in response to RNA signals.
Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , RNA de Plantas/metabolismo , Alelos , Aminoácido Oxirredutases/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Domínio Catalítico/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/fisiologia , Modelos Genéticos , Mutação , Regiões Promotoras Genéticas/genética , RNA de Plantas/genéticaRESUMO
To analyze relationships between RNA signals, DNA methylation and chromatin modifications, we performed a genetic screen to recover Arabidopsis mutants defective in RNA-directed transcriptional silencing and methylation of a nopaline synthase promoter-neomycinphosphotransferase II (NOSpro- NPTII) target gene. Mutants were identified by screening for recovery of kanamycin resistance in the presence of an unlinked silencing complex encoding NOSpro double-stranded RNA. One mutant, rts1 (RNA-mediated transcriptional silencing), displayed moderate recovery of NPTII gene expression and partial loss of methylation in the target NOSpro, predominantly at symmetrical C(N)Gs. The RTS1 gene was isolated by positional cloning and found to encode a putative histone deacetylase, HDA6. The more substantial decrease in methylation of symmetrical compared with asymmetrical cytosines in rts1 mutants suggests that HDA6 is dispensable for RNA-directed de novo methylation, which results in intermediate methylation of cytosines in all sequence contexts, but is necessary for reinforcing primarily C(N)G methylation induced by RNA. Because CG methylation in centromeric and rDNA repeats was not reduced in rts1 mutants, HDA6 might be specialized for the RNA- directed pathway of genome modification.
Assuntos
Metilação de DNA , Histona Desacetilases/metabolismo , Histona Desacetilases/fisiologia , RNA de Cadeia Dupla , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis , Mapeamento Cromossômico , Clonagem Molecular , Inativação Gênica , Teste de Complementação Genética , Homozigoto , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação , Plantas Geneticamente Modificadas , Ribonucleases/metabolismo , Sulfitos/farmacologia , Transcrição Gênica , TransgenesRESUMO
In plants, double-stranded RNA that is processed to short RNAs approximately 21-24 nt in length can trigger two types of epigenetic gene silencing. Posttranscriptional gene silencing, which is related to RNA interference in animals and quelling in fungi, involves targeted elimination of homologous mRNA in the cytoplasm. RNA-directed DNA methylation involves de novo methylation of almost all cytosine residues within a region of RNA-DNA sequence identity. RNA-directed DNA methylation is presumed to be responsible for the methylation observed in protein coding regions of posttranscriptionally silenced genes. Moreover, a type of transcriptional gene silencing and de novo methylation of homologous promoters in trans can occur if a double-stranded RNA contains promoter sequences. Although RNA-directed DNA methylation has been described so far only in plants, there is increasing evidence that RNA can also target genome modifications in other organisms. To understand how RNA directs methylation to identical DNA sequences and how changes in chromatin configuration contribute to initiating or maintaining DNA methylation induced by RNA, a promoter double-stranded RNA-mediated transcriptional gene silencing system has been established in Arabidopsis. A genetic analysis of this system is helping to unravel the relationships among RNA signals, DNA methylation, and chromatin structure.
Assuntos
Arabidopsis/genética , Metilação de DNA , RNA de Plantas/fisiologia , Sequência de Bases , DNA de Plantas , Dados de Sequência Molecular , Regiões Promotoras Genéticas , TransgenesRESUMO
The Arabidopsis genome encodes four Dicer-like (DCL) proteins, two of which contain putative nuclear localization signals. This suggests one or more nuclear pathways for processing double-stranded (ds) RNA in plants. To study the subcellular location of processing of nuclear-encoded dsRNA involved in transcriptional silencing, we examined short interfering (si) RNA and micro (mi) RNA accumulation in transgenic Arabidopsis expressing nuclear and cytoplasmic variants of P19, a viral protein that suppresses posttranscriptional gene silencing. P19 binds specifically to DCL-generated 21- to 25-nucleotide (nt) dsRNAs with 2-nt 3' overhangs and reportedly suppresses the accumulation of all size classes of siRNA. Nuclear P19 resulted in a significant reduction of 21- to 22-nt siRNAs and a 21-nt miRNA, but had a lesser effect on 24-nt siRNAs. Cytoplasmic P19 did not decrease the quantity but resulted in a 2-nt truncation of siRNAs and miRNA. This suggests that the direct products of DCL cleavage of dsRNA precursors of 21- to 22-nt siRNAs and miRNA are present in the nucleus, where their accumulation is partially repressed, and in the cytoplasm, where both normal sized and truncated forms accumulate. DCL1, which contains two putative nuclear localization signals, is required for miRNA production but not siRNA production. DCL1-green fluorescent protein fusion proteins localize to nuclei in transient expression assays, indicating that DCL1 is a nuclear protein. The results are consistent with a model in which dsRNA precursors of miRNAs and at least some 21- to 22-nt siRNAs are processed in the nucleus, the former by nuclear DCL1 and the latter by an unknown nuclear DCL.
Assuntos
Arabidopsis/genética , Núcleo Celular/metabolismo , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Sequência de Bases , Núcleo Celular/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
To test the influence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in N. sylvestris and in N. sylvestris-N. tomentosiformis hybrids, the R8.3 sequence was placed upstream of a nopaline synthase promoter (NOSpro)-NPTII reporter gene in a T-DNA construct. A number of transgenic N. sylvestris lines were produced and in most, the NPTII gene was expressed. In one line, however, the NPTII gene became silenced and methylated in the NOSpro region. The silenced locus was able to trans-inactivate and induce methylation of two stably expressed transgene loci comprising a similar construct. Nucleotide sequence analyses of the three transgene loci revealed that they each contained a single incomplete copy of the T-DNA, which had sustained deletions of varying sizes in the R8.3 region. Paradoxically, the R8.3 DNA upstream of the two active, unmethylated NOSpro-NPTII genes was highly methylated, whereas the R8.3 DNA upstream of the silenced, methylated NOSpro-NPTII gene was less methylated. The methylated portions of the R8.3 sequence corresponded to retroelement remnants. An active NOSpro-NPTII gene downstream of a nearly intact R8.3 sequence did not become methylated in N. sylvestris-N. tomentosiformis hybrids. Thus, methylation in the R8.3 sequence did not spread into adjoining transgene promoters and the effect of the R8.3 dispersed repeat family on transgene expression was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in the context of other possible triggers of silencing.