BACKGROUND: Methylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert. RESULTS: We show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases. CONCLUSIONS: Our results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.

• Keywords
• Epigenetics, RNA interference, RdDM, Transcriptional gene silencing, sRNA sequencing,
• MeSH
• Caulimovirus genetics   MeSH
• Estradiol pharmacology   MeSH
• RNA, Small Interfering genetics   metabolism   MeSH
• DNA Methylation * drug effects   MeSH
• Plasmids genetics   metabolism   MeSH
• Promoter Regions, Genetic MeSH
• RNA Interference MeSH
• Plant Cells metabolism   MeSH
• Nicotiana cytology   MeSH
• Green Fluorescent Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Estradiol MeSH
• RNA, Small Interfering MeSH
• Green Fluorescent Proteins MeSH

Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.

• Keywords
• 5-Azacytidine, De novo regeneration, Methylation, Reactivation, TGS, Transgene silencing,
• MeSH
• Azacitidine pharmacology   MeSH
• Plants, Genetically Modified drug effects   genetics   metabolism   MeSH
• DNA Methylation drug effects   genetics   MeSH
• Gene Expression Regulation, Plant drug effects   genetics   MeSH
• Solanum tuberosum drug effects   genetics   metabolism   MeSH
• Transgenes drug effects   genetics   MeSH
• Gene Silencing MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Azacitidine MeSH
• Green Fluorescent Proteins MeSH

In plants, silencing is usually accompanied by DNA methylation and heterochromatic histone marks. We studied these epigenetic modifications in different epialleles of 35S promoter (P35S)-driven tobacco transgenes. In locus 1, the T-DNA was organized as an inverted repeat, and the residing neomycin phosphotransferase II reporter gene (P35S-nptII) was silenced at the posttranscriptional (PTGS) level. Transcriptionally silenced (TGS) epialleles were generated by trans-acting RNA signals in hybrids or in a callus culture. PTGS to TGS conversion in callus culture was accompanied by loss of the euchromatic H3K4me3 mark in the transcribed region of locus 1, but this change was not transmitted to the regenerated plants from these calli. In contrast, cytosine methylation that spread from the transcribed region into the promoter was maintained in regenerants. Also, the TGS epialleles generated by trans-acting siRNAs did not change their active histone modifications. Thus, both TGS and PTGS epialleles exhibit euchromatic (H3K4me3 and H3K9ac) histone modifications despite heavy DNA methylation in the promoter and transcribed region, respectively. However, in the TGS locus (271), abundant heterochromatic H3K9me2 marks and DNA methylation were present on P35S. Heterochromatic histone modifications are not automatically installed on transcriptionally silenced loci in tobacco, suggesting that repressive histone marks and cytosine methylation may be uncoupled. However, transient loss of euchromatic modifications may guide de novo DNA methylation leading to formation of stable repressed epialleles with recovered eukaryotic marks. Compilation of available data on epigenetic modification of inactivated P35S in different systems is provided.

• Keywords
• DNA methylation, callus, dedifferentiation, histone modification, tobacco, transgene silencing,
• MeSH
• Chromatin genetics   metabolism   MeSH
• Epigenesis, Genetic * MeSH
• Plants, Genetically Modified genetics   metabolism   MeSH
• Histones genetics   metabolism   MeSH
• Bony Callus metabolism   MeSH
• DNA Methylation MeSH
• Gene Expression Regulation, Plant MeSH
• Nicotiana genetics   metabolism   MeSH
• Transgenes MeSH
• Gene Silencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• Histones MeSH

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.

• MeSH
• Alleles MeSH
• Epigenesis, Genetic MeSH
• Transcription, Genetic MeSH
• Plants, Genetically Modified genetics   MeSH
• Genomic Imprinting MeSH
• RNA, Small Interfering genetics   MeSH
• DNA Methylation * MeSH
• Promoter Regions, Genetic MeSH
• Gene Expression Regulation, Plant * MeSH
• RNA Interference MeSH
• Nicotiana genetics   MeSH
• Transgenes genetics   MeSH
• Silencer Elements, Transcriptional genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Small Interfering MeSH

BACKGROUND AND AIMS: Transgenic plants represent an excellent tool for experimental plant biology and are an important component of modern agriculture. Fully understanding the stability of transgene expression is critical in this regard. Most changes in transgene expression occur soon after transformation and thus unwanted lines can be discarded easily; however, transgenes can be silenced long after their integration. METHODS: To study the long-term changes in transgene expression in potato (Solanum tuberosum), the activity of two reporter genes, encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII), was monitored in a set of 17 transgenic lines over 5 years of vegetative propagation in vitro. KEY RESULTS: A decrease in transgene expression was observed mainly in lines with higher initial GFP expression and a greater number of T-DNA insertions. Complete silencing of the reporter genes was observed in four lines (nearly 25 %), all of which successively silenced the two reporter genes, indicating an interconnection between their silencing. The loss of GFP fluorescence always preceded the loss of kanamycin resistance. Treatment with the demethylation drug 5-azacytidine indicated that silencing of the NPTII gene, but probably not of GFP, occurred directly at the transcriptional level. Successive silencing of the two reporter genes was also reproduced in lines with reactivated expression of previously silenced transgenes. CONCLUSIONS: We suggest a hypothetical mechanism involving the successive silencing of the two reporter genes that involves the switch of GFP silencing from the post-transcriptional to transcriptional level and subsequent spreading of methylation to the NPTII gene.

• MeSH
• Plants, Genetically Modified genetics   growth & development   MeSH
• Kanamycin Kinase genetics   MeSH
• DNA Methylation MeSH
• Gene Expression Regulation, Plant genetics   MeSH
• Solanum tuberosum genetics   growth & development   MeSH
• Transgenes genetics   MeSH
• Gene Silencing physiology   MeSH
• Green Fluorescent Proteins genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Kanamycin Kinase MeSH
• Green Fluorescent Proteins MeSH

BACKGROUND: Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. RESULTS: The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. CONCLUSION: The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

• MeSH
• Clone Cells MeSH
• Cell Culture Techniques MeSH
• Cell Line MeSH
• Plants, Genetically Modified genetics   MeSH
• Gene Expression Regulation, Plant * MeSH
• Nicotiana genetics   MeSH
• Gene Transfer Techniques * MeSH
• Transformation, Genetic MeSH
• Transgenes * MeSH
• Green Fluorescent Proteins genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Green Fluorescent Proteins MeSH

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.

• MeSH
• Alleles * MeSH
• Cell Culture Techniques MeSH
• Epigenesis, Genetic * MeSH
• Plants, Genetically Modified MeSH
• DNA Methylation MeSH
• Inverted Repeat Sequences genetics   MeSH
• Open Reading Frames genetics   MeSH
• Cellular Reprogramming genetics   MeSH
• Promoter Regions, Genetic genetics   MeSH
• Regeneration genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• RNA, Plant metabolism   MeSH
• Sequence Analysis, DNA MeSH
• Sulfites MeSH
• Blotting, Southern MeSH
• Nicotiana genetics   physiology   MeSH
• Transgenes * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• hydrogen sulfite MeSH Browser
• RNA, Plant MeSH
• Sulfites MeSH

The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the evolution of an IFN-resistant state in vitro using melanoma cell lines. We found that the cells became less sensitive to antiproliferative effect of IFN-gamma after prolonged cultivation enabling us to isolate sensitive and resistant subclones of the parental line. We investigated transcription of signal transducer and activator of transcription (STAT) 1-6 and suppressor of cytokine signalling (SOCS) 1-3 genes, and phosphorylation of STAT 1 protein. The resistant subline (termed WM 1158R) differed from the sensitive subline (WM 1158S) by a constitutive expression of SOCS 3, lack or weak SOCS 1-3 activation following IFN-gamma, and short duration of cytokine activatory signal. Similar correlations were observed in additional melanoma lines differing in IFN sensitivities. At the protein level, IFN-gamma induced strong and prolonged STAT 1 activation at serine 727 (S727) in WM 1158R while in WM 1158S cells phosphorylation of this amino acid was much less pronounced. On the other hand, phosphorylation of tyrosine 701 (Y701) was stimulated regardless of the sensitivity phenotype. In conclusion, constitutive expression of SOCS 3 is correlated with attenuation of its induction following IFN treatment. These results suggest that progression of melanoma cells from IFN sensitivity to IFN insensitivity associates with changes in SOCS expression.

• MeSH
• Drug Resistance, Neoplasm genetics   MeSH
• Gene Expression MeSH
• Phosphorylation MeSH
• Interferon-gamma pharmacology   therapeutic use   MeSH
• Humans MeSH
• Melanoma drug therapy   genetics   MeSH
• Cell Line, Tumor MeSH
• Skin Neoplasms drug therapy   genetics   MeSH
• Cell Proliferation drug effects   MeSH
• Suppressor of Cytokine Signaling 3 Protein MeSH
• Suppressor of Cytokine Signaling Proteins genetics   MeSH
• Gene Expression Regulation, Neoplastic * MeSH
• Gene Expression Profiling MeSH
• STAT1 Transcription Factor metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Interferon-gamma MeSH
• Suppressor of Cytokine Signaling 3 Protein MeSH
• Suppressor of Cytokine Signaling Proteins MeSH
• SOCS3 protein, human MeSH Browser
• STAT1 protein, human MeSH Browser
• STAT1 Transcription Factor MeSH

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.

• MeSH
• Alleles MeSH
• Epigenesis, Genetic * MeSH
• Plants, Genetically Modified genetics   metabolism   MeSH
• Kanamycin Kinase genetics   metabolism   MeSH
• DNA Methylation MeSH
• RNA, Untranslated analysis   MeSH
• Repetitive Sequences, Nucleic Acid * MeSH
• Genes, Reporter MeSH
• Nicotiana genetics   MeSH
• Transgenes * MeSH
• Gene Silencing * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Kanamycin Kinase MeSH
• RNA, Untranslated MeSH

Epigenetic changes accompanying plant cell dedifferentiation and differentiation are reported in 35S ribosomal DNA (rDNA) of tobacco (Nicotiana tabacum). There was a reduction of CG and CNG methylation in both intergenic and genic regions of the rDNA cistron in fully dedifferentiated callus and root compared to leaf. The rDNA hypomethylation was not random, but targeted to particular rDNA gene families at units that are clustered within the tandem array. The process of hypomethylation was initiated as early as 2 weeks after the callus induction and established epigenetic patterns were stably maintained throughout prolonged culture. However, regenerated plants and their progeny showed partial and complete remethylation of units, respectively. Nuclear run-on assays revealed a 2-fold increase of primary (unprocessed) ribosomal RNA transcripts in callus compared to leaf tissue. However, the abundance of mature transcripts in callus was elevated by only about 25%. Fluorescence in situ hybridization analysis of interphase nuclei showed high levels of rDNA chromatin condensation in both callus and leaf, with substantially less decondensed rDNA than is observed in meristematic root-tip cells. It is likely that the regions of the rDNA locus showing decondensation correspond to the clusters of hypomethylated units that occur in the tandem array at each locus. The data together indicate that the establishment of pluripotency and cell proliferation occurring with callus induction is associated with enhanced ribosomal RNA gene expression and overall rDNA hypomethylation, but is not associated with material-enhanced relaxation of chromatin structure (decondensation) at rDNA loci.

• MeSH
• Cell Differentiation * MeSH
• Chromatin chemistry   metabolism   MeSH
• Transcription, Genetic genetics   MeSH
• In Situ Hybridization, Fluorescence MeSH
• Interphase MeSH
• Plant Roots genetics   MeSH
• Cells, Cultured MeSH
• Plant Leaves cytology   genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• DNA Methylation * MeSH
• Regeneration MeSH
• Gene Expression Regulation, Plant MeSH
• RNA, Ribosomal genetics   MeSH
• Nicotiana cytology   genetics   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• RNA, Messenger MeSH
• RNA, Ribosomal MeSH