RESUMO
Hydroponic experiments were performed to examine the effect of prolonged sulfate limitation combined with cadmium (Cd) exposure in Arabidopsis thaliana and a potential Cd hyperaccumulator, Nicotiana tabacum. Low sulfate treatments (20 and 40 µM MgSO4) and Cd stress (4 µM CdCl2) showed adverse effects on morphology, photosynthetic and biochemical parameters and the nutritional status of both species. For example, Cd stress decreased NO3- root content under 20 µM MgSO4 to approximately 50% compared with respective controls. Interestingly, changes in many measured parameters, such as chlorophyll and carotenoid contents, the concentrations of anions, nutrients and Cd, induced by low sulfate supply, Cd exposure or a combination of both factors, were species-specific. Our data showed opposing effects of Cd exposure on Ca, Fe, Mn, Cu and Zn levels in roots of the studied plants. In A. thaliana, levels of glutathione, phytochelatins and glucosinolates demonstrated their distinct involvement in response to sub-optimal growth conditions and Cd stress. In shoot, the levels of phytochelatins and glucosinolates in the organic sulfur fraction were not dependent on sulfate supply under Cd stress. Altogether, our data showed both common and species-specific features of the complex plant response to prolonged sulfate deprivation and/or Cd exposure.
Assuntos
Arabidopsis , Fitoquelatinas , Cádmio/toxicidade , Nicotiana , Sulfatos/farmacologia , Glucosinolatos/farmacologia , Nutrientes , Suplementos Nutricionais , Raízes de PlantasRESUMO
Reshaping of the chromatin landscape under oxidative stress is of paramount importance for mounting an effective stress response. Unbiased systemic identification and quantification of histone marks is crucial for understanding the epigenetic component of plant responses to adverse environmental conditions. We describe a detailed method for isolation of plant histones and subsequent bottom-up proteomics approach for characterization of acetylation and methylation status. By performing label-free quantitative mass spectrometry analysis, relative abundances of histone marks can be statistically compared between experimental conditions.
Assuntos
Histonas , Processamento de Proteína Pós-Traducional , Acetilação , Código das Histonas , Histonas/metabolismo , MetilaçãoRESUMO
Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are crucial for the maintenance of genome integrity. In most plants, telomeres consist of conserved tandem repeat units comprising the TTTAGGG motif. Recently, non-canonical telomeres were described in several plants and plant taxons, including the carnivorous plant Genlisea hispidula (TTCAGG/TTTCAGG), the genus Cestrum (Solanaceae; TTTTTTAGGG), and plants from the Asparagales order with either a vertebrate-type telomere repeat TTAGGG or Allium genus-specific CTCGGTTATGGG repeat. We analyzed epigenetic modifications of telomeric histones in plants with canonical and non-canonical telomeres, and further in telomeric chromatin captured from leaves of Nicotiana benthamiana transiently transformed by telomere CRISPR-dCas9-eGFP, and of Arabidopsis thaliana stably transformed with TALE_telo C-3×GFP. Two combinatorial patterns of telomeric histone modifications were identified: (i) an Arabidopsis-like pattern (A. thaliana, G. hispidula, Genlisea nigrocaulis, Allium cepa, Narcissus pseudonarcissus, Petunia hybrida, Solanum tuberosum, Solanum lycopersicum) with telomeric histones decorated predominantly by H3K9me2; (ii) a tobacco-like pattern (Nicotiana tabacum, N. benthamiana, C. elegans) with a strong H3K27me3 signal. Our data suggest that epigenetic modifications of plant telomere-associated histones are related neither to the sequence of the telomere motif nor to the lengths of the telomeres. Nor the phylogenetic position of the species plays the role; representatives of the Solanaceae family are included in both groups. As both patterns of histone marks are compatible with fully functional telomeres in respective plants, we conclude that the described specific differences in histone marks are not critical for telomere functions.
Assuntos
Epigenômica , Código das Histonas/genética , Plantas/genética , Telômero/genética , Arabidopsis/genética , Cromatina/genética , Filogenia , Nicotiana/genéticaRESUMO
Telomerase is essential for the maintenance of telomeres, structures located at the ends of linear eukaryotic chromosomes that are crucial for genomic stability. Telomerase has been frequently explored in mammals because of its activity in many types of cancers, but knowledge in plants is rather sketchy despite plants representing useful models due to peculiarities in their telomeres and telomerase biology. We studied in planta complementation of telomerase in Arabidopsis thaliana mutant plants with disrupted expression of the gene encoding the telomerase protein subunit (AtTERT) and significantly shortened telomeres. We found that the upstream region of AtTERT, previously identified as a putative minimal promoter, was essential for reconstitution of telomerase function, as demonstrated by the full or partial recovery of the telomere phenotype in mutants. In contrast, transformation by the full length AtTERT gene construct resulted in more progressive telomere shortening in mutants and even in wild type plants, despite the high level of AtTERT transcript and telomerase activity detected by in vitro assay. Thus, the telomerase protein subunit putative promoter is essential for in planta telomerase reconstitution and restoration of its catalytical activity. Contributions from other factors, including those tissue-specific, for proper telomerase function are discussed.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Telomerase/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regiões Promotoras Genéticas/genética , Telomerase/genéticaRESUMO
Parallel research on multiple model organisms shows that while some principles of telomere biology are conserved among all eukaryotic kingdoms, we also find some deviations that reflect different evolutionary paths and life strategies, which may have diversified after the establishment of telomerase as a primary mechanism for telomere maintenance. Much more than animals, plants have to cope with environmental stressors, including genotoxic factors, due to their sessile lifestyle. This is, in principle, made possible by an increased capacity and efficiency of the molecular systems ensuring maintenance of genome stability, as well as a higher tolerance to genome instability. Furthermore, plant ontogenesis differs from that of animals in which tissue differentiation and telomerase silencing occur during early embryonic development, and the "telomere clock" in somatic cells may act as a preventive measure against carcinogenesis. This does not happen in plants, where growth and ontogenesis occur through the serial division of apical meristems consisting of a small group of stem cells that generate a linear series of cells, which differentiate into an array of cell types that make a shoot and root. Flowers, as generative plant organs, initiate from the shoot apical meristem in mature plants which is incompatible with the human-like developmental telomere shortening. In this review, we discuss differences between human and plant telomere biology and the implications for aging, genome stability, and cell and organism survival. In particular, we provide a comprehensive comparative overview of telomere proteins acting in humans and in Arabidopsis thaliana model plant, and discuss distinct epigenetic features of telomeric chromatin in these species.
Assuntos
Plantas/metabolismo , Telômero/metabolismo , Senescência Celular/genética , Cromatina/metabolismo , Epigênese Genética , Humanos , Telomerase/metabolismoRESUMO
KEY MESSAGE: Standard pathways involved in the regulation of telomere stability do not contribute to gradual telomere elongation observed in the course of A. thaliana calli propagation. Genetic and epigenetic changes accompanying the culturing of plant cells have frequently been reported. Here we aimed to characterize the telomere homeostasis during long term callus propagation. While in Arabidopsis thaliana calli gradual telomere elongation was observed, telomeres were stable in Nicotiana tabacum and N. sylvestris cultures. Telomere elongation during callus propagation is thus not a general feature of plant cells. The long telomere phenotype in Arabidopsis calli was correlated neither with changes in telomerase activity nor with activation of alternative mechanisms of telomere elongation. The dynamics of telomere length changes was maintained in mutant calli with loss of function of important epigenetic modifiers but compromised in the presence of epigenetically active drug zebularine. To examine whether the cell culture-induced disruption of telomere homeostasis is associated with the modulated structure of chromosome ends, epigenetic properties of telomere chromatin were analysed. Albeit distinct changes in epigenetic modifications of telomere histones were observed, these were broadly stochastic. Our results show that contrary to animal cells, the structure and function of plant telomeres is not determined significantly by the epigenetic character of telomere chromatin. Set of differentially transcribed genes was identified in calli, but considering the known telomere- or telomerase-related functions of respective proteins, none of these changes per se was apparently related to the elongated telomere phenotype. Based on our data, we propose that the disruption in telomere homeostasis in Arabidopsis calli arises from the interplay of multiple factors, as a part of reprogramming of plant cells to long-term culture conditions.
Assuntos
Arabidopsis/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Técnicas de Cultura de Tecidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Citidina/análogos & derivados , Citidina/farmacologia , Ecótipo , Epigênese Genética/efeitos dos fármacos , Genes de Plantas , Histonas/metabolismo , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração/efeitos dos fármacos , Especificidade da Espécie , Telomerase/metabolismo , Homeostase do Telômero/efeitos dos fármacos , Nicotiana/genéticaAssuntos
Antígenos CD20/metabolismo , Antineoplásicos Imunológicos/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Rituximab/farmacologia , Transdução de Sinais/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Fenótipo , Rituximab/uso terapêutico , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
MAIN CONCLUSION: In tobacco, three sequence variants of the TERT gene have been described. We revealed unbalanced levels of TERT variant transcripts in vegetative tobacco tissues and enhanced TERT transcription and telomerase activity in reproductive tissues. Telomerase is a ribonucleoprotein complex responsible for the maintenance of telomeres, structures delimiting ends of linear eukaryotic chromosomes. In the Nicotiana tabacum (tobacco) allotetraploid plant, three sequence variants (paralogs) of the gene coding for the telomerase reverse transcriptase subunit (TERT) have been described, two of them derived from the maternal N. sylvestris genome (TERT_Cs, TERT_D) and one originated from the N. tomentosiformis paternal genome (TERT_Ct). In this work, we analyzed the transcription of TERT variants in correlation with telomerase activity in tobacco tissues. High and approximately comparable levels of TERT_Ct and TERT_Cs transcripts were detected in seedlings, roots, flower buds and leaves, while the transcript of the TERT_D variant was markedly underrepresented. Similarly, in N. sylvestris tissues, TERT_Cs transcript significantly predominated. A specific pattern of TERT transcripts was found in samples of tobacco pollen with the TERT_Cs variant clearly dominating particularly at the early stage of pollen development. Detailed analysis of TERT_C variants representation in functionally distinct fractions of pollen transcriptome revealed their prevalence in large ribonucleoprotein particles encompassing translationally silent mRNA; only a minority of TERT_Ct and TERT_Cs transcripts were localized in actively translated polysomes. Histones of the TERT_C chromatin were decorated predominantly with the euchromatin-specific epigenetic modification in both telomerase-positive and telomerase-negative tobacco tissues. We conclude that the existence and transcription pattern of tobacco TERT paralogs represents an interesting phenomenon and our results indicate its functional significance. Nicotiana species have again proved to be appropriate and useful model plants in telomere biology studies.
Assuntos
Regulação da Expressão Gênica de Plantas , Variação Genética , Nicotiana/genética , Especificidade de Órgãos/genética , Telomerase/genética , Núcleo Celular/genética , Imunoprecipitação da Cromatina , Eucromatina/metabolismo , Histonas/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Polirribossomos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/metabolismo , Transcrição GênicaRESUMO
Telomeres and genes encoding 45S ribosomal RNA (rDNA) are frequently located adjacent to each other on eukaryotic chromosomes. Although their primary roles are different, they show striking similarities with respect to their features and additional functions. Both genome domains have remarkably dynamic chromatin structures. Both are hypersensitive to dysfunctional histone chaperones, responding at the genomic and epigenomic levels. Both generate non-coding transcripts that, in addition to their epigenetic roles, may induce gross chromosomal rearrangements. Both give rise to chromosomal fragile sites, as their replication is intrinsically problematic. However, at the same time, both are essential for maintenance of genomic stability and integrity. Here we discuss the structural and functional inter-connectivity of telomeres and rDNA, with a focus on recent results obtained in plants.
Assuntos
Cromatina/química , DNA Ribossômico/metabolismo , Plantas/genética , Telômero/metabolismo , Cromatina/metabolismo , Replicação do DNA , DNA de Plantas/química , DNA de Plantas/metabolismo , DNA Ribossômico/química , Epigênese Genética , Instabilidade Genômica , Histonas/genética , Histonas/metabolismo , Telômero/genéticaRESUMO
Epigenetic mechanisms are involved in regulation of crucial cellular processes in eukaryotic organisms. Data on the epigenetic features of plant telomeres and their epigenetic regulation were published mostly for Arabidopsis thaliana, in which the presence of interstitial telomeric repeats (ITRs) may interfere with genuine telomeres in most analyses. Here, we studied the epigenetic landscape and transcription of telomeres and ITRs in Nicotiana tabacum with long telomeres and no detectable ITRs, and in Ballantinia antipoda with large blocks of pericentromeric ITRs and relatively short telomeres. Chromatin of genuine telomeres displayed heterochromatic as well as euchromatic marks, while ITRs were just heterochromatic. Methylated cytosines were present at telomeres and ITRs, but showed a bias with more methylation toward distal telomere positions and different blocks of B. antipoda ITRs methylated to different levels. Telomeric transcripts TERRA (G-rich) and ARRET (C-rich) were identified in both plants and their levels varied among tissues with a maximum in blossoms. Plants with substantially different proportions of internally and terminally located telomeric repeats are instrumental in clarifying the chromatin status of telomeric repeats at distinct chromosome locations.
RESUMO
Although telomerase (EC 2.7.7.49) is important for genome stability and totipotency of plant cells, the principles of its regulation are not well understood. Therefore, we studied subcellular localization and function of the full-length and truncated variants of the catalytic subunit of Arabidopsis thaliana telomerase, AtTERT, in planta. Our results show that multiple sites in AtTERT may serve as nuclear localization signals, as all the studied individual domains of the AtTERT were targeted to the nucleus and/or the nucleolus. Although the introduced genomic or cDNA AtTERT transgenes display expression at transcript and protein levels, they are not able to fully complement the lack of telomerase functions in tert -/- mutants. The failure to reconstitute telomerase function in planta suggests a more complex telomerase regulation in plant cells than would be expected based on results of similar experiments in mammalian model systems.
Assuntos
Arabidopsis/genética , Telomerase/química , Telomerase/genética , Telomerase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Domínio Catalítico/genética , Nucléolo Celular/enzimologia , Nucléolo Celular/genética , Núcleo Celular/enzimologia , Núcleo Celular/genética , Regulação da Expressão Gênica de Plantas , Sinais de Localização Nuclear/genética , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Splicing de RNA , Relação Estrutura-Atividade , Nicotiana/genéticaRESUMO
Apoptotic bodies are the most condensed form of chromatin. In general, chromatin structure and function are mostly dictated by histone post-translational modifications. Thus, we have analyzed the histone signature in apoptotic cells, characterized by pronounced chromatin condensation. Here, H2B mono-acetylation, and H3K9 and H4 acetylation was significantly decreased in apoptotic cells, which maintained a high level of H3K9 methylation. This phenotype was independent of p53 function and distinct levels of anti-apoptotic Bcl2 protein. Interestingly, after etoposide treatment of leukemia and multiple myeloma cells, H3K9 and H4 hypoacetylation was accompanied by increased H3K9me2, but not H3K9me1 or H3K9me3. In adherent mouse fibroblasts, a high level of H3K9me3 and histone deacetylation in apoptotic bodies was likely responsible for the pronounced (â¼40%) recovery of GFP-HP1α and GFP-HP1ß after photobleaching. HP1 mobility in apoptotic cells appeared to be unique because limited exchange after photobleaching was observed for other epigenetically important proteins, including GFP-JMJD2b histone demethylase (â¼10% fluorescence recovery) or Polycomb group-related GFP-BMI1 protein (â¼20% fluorescence recovery). These findings imply a novel fact that only certain subset of proteins in apoptotic bodies is dynamic.
Assuntos
Apoptose/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Proteínas Cromossômicas não Histona/genética , Epigênese Genética/efeitos dos fármacos , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetilação , Animais , Antineoplásicos Fitogênicos , Adesão Celular , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Etoposídeo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Histonas/genética , Humanos , Cinética , Metilação , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Telomerase is essential for proper functioning of telomeres in eukaryotes. We cloned and characterised genes for the protein subunit of telomerase (TERT) in the allotetraploid Nicotiana tabacum (tobacco) and its diploid progenitor species Nicotiana sylvestris and Nicotiana tomentosiformis with the aim of determining if allopolyploidy (hybridisation and genome duplication) influences TERT activity and divergence. Two of the three sequence variants present in the tobacco genome (NtTERT-C/s and NtTERT-D) revealed similarity to two sequence variants found in N. sylvestris and another variant (NtTERT-C/t) was similar to TERT of N. tomentosiformis. Variants of N. sylvestris origin showed less similarity to each other (80.5 % in the genomic region; 90.1 % in the coding sequence) than that between the NtTERT-C/s and NtTERT-C/t variants (93.6 and 97.2 %, respectively). The NtTERT-D variant was truncated at the 5' end, and indels indicated that it was a pseudogene. All tobacco variants were transcribed and alternatively spliced sequences were detected. Analysis of gene arrangements uncovered a novel exon in the N-terminal domain of TERT variants, a feature that is likely to be commonly found in Solanaceae species. In addition, species-specific duplications were observed within exon 5. The putative function, copy number and evolutionary origin of these NtTERT sequence variants are discussed.
Assuntos
Nicotiana/genética , Telomerase/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Evolução Molecular , Éxons , Ordem dos Genes , Rearranjo Gênico , Loci Gênicos , Genoma de Planta , Íntrons , Dados de Sequência Molecular , Pseudogenes , Isoformas de RNA , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Transcrição GênicaRESUMO
Telomere homeostasis is regulated at multiple levels, including the local chromatin structure of telomeres and subtelomeres. Recent reports demonstrated that a decrease in repressive chromatin marks, such as levels of cytosine methylation in subtelomeric regions, results in telomere elongation in mouse cells. Here we show that a considerable fraction of cytosines is methylated not only in subtelomeric, but also in telomeric DNA of tobacco BY-2 cells. Drug-induced hypomethylation (demonstrated at subtelomeric, telomeric, and global DNA levels) results in activation of telomerase. However, in contrast to mouse cells, the decrease in 5-methylcytosine levels and upregulation of telomerase do not result in any changes of telomere lengths. These results demonstrate the involvement of epigenetic mechanisms in the multilevel process of regulation of telomerase activity in plant cells and, at the same time, they indicate that changes in telomerase activity can be overridden by other factors governing telomere length stability.
Assuntos
Adenina/análogos & derivados , Citidina/análogos & derivados , DNA de Plantas/efeitos dos fármacos , Nicotiana/efeitos dos fármacos , Telomerase/metabolismo , Telômero/efeitos dos fármacos , Adenina/farmacologia , Células Cultivadas , Citidina/farmacologia , Metilação de DNA/efeitos dos fármacos , DNA de Plantas/química , Ativação Enzimática/efeitos dos fármacos , Epigênese Genética , Nucleossomos/efeitos dos fármacos , Nucleossomos/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Telômero/química , Telômero/metabolismo , Nicotiana/citologia , Nicotiana/genética , Nicotiana/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica de Plantas , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Elementos Silenciadores Transcricionais/genética , Alelos , Epigênese Genética , Impressão Genômica , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/genética , Transcrição Gênica , Transgenes/genéticaRESUMO
Telomeres are specialized chromatin structures that are situated at the end of linear chromosomes and play an important role in cell senescence and immortalization. Here, we investigated whether changes in histone signature influence the nuclear arrangement and positioning of telomeres. Analysis of mouse embryonic fibroblasts revealed that telomeres were organized into specific clusters that partially associated with centromeric clusters. This nuclear arrangement was influenced by deficiency of the histone methyltransferase SUV39h, LMNA deficiency, and the histone deacetylase inhibitor Trichostatin A (TSA). Similarly, nuclear radial distributions of telomeric clusters were preferentially influenced by TSA, which caused relocation of telomeres closer to the nuclear center. Telomeres also co-localized with promyelocytic leukemia bodies (PML). This association was increased by SUV39h deficiency and decreased by LMNA deficiency. These differences could be explained by differing levels of the telomerase subunit, TERT, in SUV39h- and LMNA-deficient fibroblasts. Taken together, our data show that SUV39h and A-type lamins likely play a key role in telomere maintenance and telomere nuclear architecture.
Assuntos
Rearranjo Gênico , Lamina Tipo A/metabolismo , Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Telômero/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Camundongos , Transporte Proteico , Complexo Shelterina , Telomerase/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Using a two-component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus 1 (Lo1) and a homologous single-copy transgene locus 2 (Lo2), we have studied the stability of the methylation patterns and trans-silencing interactions in cell culture and regenerated tobacco (Nicotiana tabacum) plants. The posttranscriptionally silenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line), while its transcriptionally silenced variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in early Lo1E/Lo2 callus, decreased transgene expression and partial loss of Lo1E promoter methylation compared with leaf tissue in the parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgenes were silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the nonmethylated Lo1 promoter acquired partial methylation in later callus stages. These data indicate that a cell culture process has brought both epialleles to a similar epigenetic ground. Bisulfite sequencing of the 35S promoter within the Lo1 silencer revealed molecules with no, intermediate, and high levels of methylation, demonstrating, to our knowledge for the first time, cell-to-cell methylation diversity of callus. Regenerated plants showed high interindividual but low intraindividual epigenetic variability, indicating that the callus-induced epiallelic variants were transmitted to plants and became fixed. We propose that epigenetic changes associated with dedifferentiation might influence regulatory pathways mediated by trans-PTGS processes.
Assuntos
Alelos , Reprogramação Celular/genética , Epigênese Genética , Sequências Repetidas Invertidas/genética , Nicotiana/genética , Transgenes , Southern Blotting , Técnicas de Cultura de Células , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Fases de Leitura Aberta/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , RNA de Plantas/metabolismo , Regeneração/genética , Análise de Sequência de DNA , Sulfitos , Nicotiana/fisiologiaRESUMO
We have studied the inheritance of the epigenetic state of tobacco transgenes whose expression was post-transcriptionally silenced by an invertedly repeated silencer locus. We show that, in hybrids, the coding region of the target neomycin phosphotransferase (nptII) gene was almost exclusively methylated at CG configurations, and dense non-CG methylation occurred in the 3' untranslated region. Homologous sequences in the silencer locus were heavily methylated at both CG and non-CG motifs. After segregation of the silencer locus, the CG methylation but not the non-CG methylation of the target genes was transmitted to the progeny. In the segregants, we observed an overall increase of CG methylation in the target genes, associated with a re-distribution from the 3' end of the coding region towards the middle. This pattern was inherited with some fluctuation for at least two additional generations in the absence of a detectable T-DNA-derived small RNA fraction. Thus CG methylation is not cleared during meiosis and may be inherited over generations without RNA signals being present. These epi-allelic variants re-expressed the reporter gene immediately after segregation of the trigger, showing that relatively dense CG methylation (approximately 60-80%) imprinted on most of the coding region (>500 bp) did not reduce expression compared with the parental non-methylated locus. We propose that the genic CG methylation seen in euchromatic regions of the genome may originate from ancient post-transcriptional gene silencing events as a result of adventitiously produced methylation-directing RNA molecules.
Assuntos
Metilação de DNA , Padrões de Herança , Nicotiana/genética , Interferência de RNA , Transgenes , Alelos , Cruzamentos Genéticos , DNA Bacteriano/metabolismo , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Nicotiana/metabolismoRESUMO
In this paper, we have compared hypomethylating ability of classical beta-d-anomer of 5-aza-2'-deoxycytidine (5-aza-CdRf) and its alpha anomer in cell cultures. Alpha anomers of nucleosides generally exhibit low biological activity compared to their beta counterparts. It is reported that alpha anomer of 5-aza-CdRf efficiently hypomethylated genomic DNA in human T-lymphoblastoid CCRF-CEM cells. Satellite 2 and 18S rDNA were hypomethylated by alpha anomer at concentrations comparable to the beta form. However, the toxicity of the alpha anomer was 4-fold less than that of beta form. Contrast to CCRF-CEM the A549 lung carcinoma cells, possessing negligible level of methylation at repetitive loci, were highly resistant to 5-aza-CdRf treatment suggesting that global genomic methylation might be needed to mediate cytotoxic effect of the drug. Possible mechanisms of inhibition of DNA methylation by alpha anomer are discussed. In conclusion, alpha anomer of 5-aza-CdRf displaying lower host cytotoxicity than the classical beta form may be of potential use in epigenetic therapy.
Assuntos
Antimetabólitos/farmacologia , Azacitidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Southern Blotting , Western Blotting , Linhagem Celular Tumoral , Sondas de DNA , Decitabina , Humanos , IsomerismoRESUMO
We studied the in trans-silencing capacities of a transgene locus that carried the neomycin phosphotransferase II reporter gene linked to the 35S promoter in an inverted repeat (IR). This transgene locus was originally posttranscriptionally silenced but switched to a transcriptionally silenced epiallele after in vitro tissue culture. Here, we show that both epialleles were strongly methylated in the coding region and IR center. However, by genomic sequencing, we found that the 1.0 kb region around the transcription start site was heavily methylated in symmetrical and non-symmetrical contexts in transcriptionally but not in posttranscriptionally silenced epilallele. Also, the posttranscriptionally silenced epiallele could trans-silence and trans-methylate homologous transgene loci irrespective of their genomic organization. We demonstrate that this in trans-silencing was accompanied by the production of small RNA molecules. On the other hand, the transcriptionally silenced variant could neither trans-silence nor trans-methylate homologous sequences, even after being in the same genetic background for generations and meiotic cycles. Interestingly, 5-aza-2-deoxy-cytidine-induced hypomethylation could partially restore signaling from the transcriptionally silenced epiallele. These results are consistent with the hypothesis that non-transcribed highly methylated IRs are poor silencers of homologous loci at non-allelic positions even across two generations and that transcription of the inverted sequences is essential for their trans-silencing potential.