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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 MgSO

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.

ARM was identified previously as an interaction partner of the telomerase protein subunit (TERT) in Arabidopsis thaliana. To investigate the interconnection between ARM and telomerase and to identify ARM cellular functions, we analyzed a set of arm mutant lines and arm/tert double mutants. Telomere length was not affected in arm single mutant plants, in contrast to double mutants. In the second generation of homozygous arm-1/tert double mutants following the heterozygous state during the double mutant construction, telomeres shortened dramatically, even below levels in tert plants displaying severe morphological defects. Intriguingly, homozygous arm-1/tert double mutants with short telomeres grew without obvious phenotypic changes for next two generations. Then, in agreement with the onset of phenotypic changes in tert, morphological defects were timed to the 5th arm-1/tert homozygous generation. RNAseq analyses of arm-1/tert and respective single mutants displayed markedly overlapping sets of differentially expressed genes in arm-1/tert double mutant and arm-1 single mutant lines, indicating a dominant effect of the ARM mutation. RNAseq data further implied ARM involvement in circadian rhythms, responses to drugs and to biotic and abiotic stimuli. In agreement with it, we observed sensitivity of arm-1 single mutant to the heat stress during germination. Altogether, our results suggest ARM involvement in crucial cellular processes without evidencing its role in the telomerase canonical function.

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats., Graphical Abstract Graphical AbstractWe present a smart strategy of telomerase RNA (TR) identification based on its conserved type-3 RNA Pol III promoter and TR template elements. We characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. TRs are validated experimentally and show conservation of core TR structural domains. These results shed light on the evolution of a key eukaryotic non-coding RNA across more than a billion years.

While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional interplays with chromatin factors along the epigenome are just starting to emerge. Here we first report that inArabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2cisrecruitment. This study unveils a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich domains at telomeric sequences and contributes to safeguard nucleus architecture.Abstract FigureTeano et al. report that that linker histone H1 and a group of H1-related telomeric proteins interplay to selectively influence thePolycombrepressive landscape at genes and telomeric repeats inArabidopsis. These findings provide a mechanistic framework by which H1 influences the epigenome and nuclear organization in a sequence-specific manner.HighlightsH1 promotes PRC2 activity and limits accessibility at a majority of genesH1 prevents PRC2 activity at telomeric DNA sequencesPRC2 repression is achieved by restricting accessibility to TRB proteinsH1 orchestrates the spatial organization of telomeres and interstitial telomeres (ITRs)

Involvement of epigenetic mechanisms in the regulation of telomeres and transposable elements (TEs), genomic regions with the protective and potentially detrimental function, respectively, has been frequently studied. Here, we analyzed telomere lengths in Arabidopsis thaliana plants of Columbia, Landsberg erecta and Wassilevskija ecotypes exposed repeatedly to the hypomethylation drug zebularine during germination. Shorter telomeres were detected in plants growing from seedlings germinated in the presence of zebularine with a progression in telomeric phenotype across generations, relatively high inter-individual variability, and diverse responses among ecotypes. Interestingly, the extent of telomere shortening in zebularine Columbia and Wassilevskija plants corresponded to the transcriptional activation of TEs, suggesting a correlated response of these genomic elements to the zebularine treatment. Changes in lengths of telomeres and levels of TE transcripts in leaves were not always correlated with a hypomethylation of cytosines located in these regions, indicating a cytosine methylation-independent level of their regulation. These observations, including differences among ecotypes together with distinct dynamics of the reversal of the disruption of telomere homeostasis and TEs transcriptional activation, reflect a complex involvement of epigenetic processes in the regulation of crucial genomic regions. Our results further demonstrate the ability of plant cells to cope with these changes without a critical loss of the genome stability.

Background Telomeres, nucleoprotein structures comprising short tandem repeats and delimiting the ends of linear eukaryotic chromosomes, play an important role in the maintenance of genome stability. Therefore, the determination of the length of telomeres is of high importance for many studies. Over the last years, new methods for the analysis of the length of telomeres have been developed, including those based on PCR or analysis of NGS data. Despite that, terminal restriction fragment (TRF) method remains the gold standard to this day. However, this method lacks universally accepted and precise tool capable to analyse and statistically evaluate TRF results. Results To standardize the processing of TRF results, we have developed WALTER, an online toolset allowing rapid, reproducible, and user-friendly analysis including statistical evaluation of the data. Given its web-based nature, it provides an easily accessible way to analyse TRF data without any need to install additional software. Conclusions WALTER represents a major upgrade from currently available tools for the image processing of TRF scans. This toolset enables a rapid, highly reproducible, and user-friendly evaluation of almost any TRF scan including in-house statistical evaluation of the data. WALTER platform together with user manual describing the evaluation of TRF scans in detail and presenting tips and troubleshooting, as well as test data to demo the software are available at https://www.ceitec.eu/chromatin-molecular-complexes-jiri-fajkus/rg51/tab?tabId=125#WALTER and the source code at https://github.com/mlyc93/WALTER.

Background The i-motif is a tetrameric DNA structure based on the formation of hemiprotonated cytosine-cytosine (C+.C) base pairs. i-motifs are widely used in nanotechnology. In biological systems, i-motifs are involved in gene regulation and in control of genome integrity. In vivo, the i-motif forming sequences are subjects of epigenetic modifications, particularly 5-cytosine methylation. In plants, natively occurring methylation patterns lead to a complex network of C+.C, 5mC+.C and 5mC+.5mC base-pairs in the i-motif stem. The impact of complex methylation patterns (CMPs) on i-motif formation propensity is currently unknown. Methods We employed CD and UV-absorption spectroscopies, native PAGE, thermal denaturation and quantum-chemical calculations to analyse the effects of native, native-like, and non-native CMPs in the i-motif stem on the i-motif stability and pKa. Results CMPs have strong influence on i-motif stability and pKa and influence these parameters in sequence-specific manner. In contrast to a general belief, i) CMPs do not invariably stabilize the i-motif, and ii) when the CMPs do stabilize the i-motif, the extent of the stabilization depends (in a complex manner) on the number and pattern of symmetric 5mC+.5mC or asymmetric 5mC+.C base pairs in the i-motif stem. Conclusions CMPs can be effectively used to fine-tune i-motif properties. Our data support the notion of epigenetic modifications as a plausible control mechanism of i-motif formation in vivo. General Significance Our results have implications in epigenetic regulation of telomeric DNA in plants and highlight the potential and limitations of engineered patterning of cytosine methylations on the i-motif scaffold in nanotechnological applications.

The use of the anti-CD20 antibody rituximab has improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab was shown to act via various mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct apoptosis, or sensitization to chemotherapy (reviewed in ref. [1]). However, the biological function of CD20 and the reasons for the impressive activity of rituximab and other anti-CD20 antibodies remain elusive. It has been suggested, but remains controversial, whether CD20 functions as a calcium channel, couples with CD40 and MHCII, or B-cell receptor (BCR), and whether it has a role in T cell-dependent and -independent immunity [1,2,3,4]. This is also of great clinical interest for the design of combinatorial treatment of rituximab with BCR inhibitors, DNA damaging or immunomodulatory drugs, or CAR T cells. We have previously shown that microenvironmental interactions upregulate the CD20 levels on CLL cells through the CXCR4/SDF-1 axis [5]. Here we describe that higher CD20 expression has a direct role in the BCR signaling in CLL cells, and a BCR-proficient intra-clonal CLL cell subpopulation is more efficiently eliminated by rituximab in vivo due to higher CD20 levels.

A high degree of developmental plasticity enables plants to adapt to continuous, often unfavorable and unpredictable changes in their environment. At the molecular level, adaptive advantages for plants are primarily provided by epigenetic machinery including DNA methylation, histone modifications, and the activity of noncoding RNA molecules. Using a mass spectrometry-based proteomic approach, we examined the levels of acetylated histone peptide forms in Arabidopsis plants with a loss of function of histone deacetylase 6 (HDA6), and in plants germinated in the presence of HDA inhibitors trichostatin A (TSA) and sodium butyrate (NaB). Our analyses revealed particular lysine sites at histone sequences targeted by the HDA6 enzyme, and by TSA- and NaB-sensitive HDAs. Compared with plants exposed to drugs, more dramatic changes in the overall profiles of histone post-translational modifications were identified in hda6 mutants. However, loss of HDA6 was not sufficient by itself to induce hyperacetylation to the maximum degree, implying complementary activities of other HDAs. In contrast to hda6 mutants that did not exhibit any obvious phenotypic defects, the phenotypes of seedlings exposed to HDA inhibitors were markedly affected, showing that the effect of these drugs on early plant development is not limited to the modulation of histone acetylation levels.

Telomeres and ribosomal RNA genes (rDNA) are essential for cell survival and particularly sensitive to factors affecting genome stability. Here, we examine the role of RAD51 and its antagonist, RTEL1, in the moss Physcomitrella patens. In corresponding mutants, we analyse their sensitivity to DNA damage, the maintenance of telomeres and rDNA, and repair of double-stranded breaks (DSBs) induced by genotoxins with various modes of action. While the loss of RTEL1 results in rapid telomere shortening, concurrent loss of both RAD51 genes has no effect on telomere lengths. We further demonstrate here the linked arrangement of 5S and 45S rRNA genes in P. patens. The spacer between 5S and 18S rRNA genes, especially the region downstream from the transcription start site, shows conspicuous clustering of sites with a high propensity to form quadruplex (G4) structures. Copy numbers of 5S and 18S rDNA are reduced moderately in the pprtel1 mutant, and significantly in the double pprad51-1-2 mutant, with no progression during subsequent cultivation. While reductions in 45S rDNA copy numbers observed in pprtel1 and pprad51-1-2 plants apply also to 5S rDNA, changes in transcript levels are different for 45S and 5S rRNA, indicating their independent transcription by RNA polymerase I and III, respectively. The loss of SOL (Sog One-Like), a transcription factor regulating numerous genes involved in DSB repair, increases the rate of DSB repair in dividing as well as differentiated tissue, and through deactivation of G2/M cell-cycle checkpoint allows the cell-cycle progression manifested as a phenotype resistant to bleomycin.

To elucidate the molecular nature of evolutionary changes of telomeres in the plant order Asparagales, we aimed to characterize telomerase RNA subunits (TRs) in these plants. The unusually long telomere repeat unit in Allium plants (12 nt) allowed us to identify TRs in transcriptomic data of representative species of the Allium genus. Orthologous TRs were then identified in Asparagales plants harbouring telomere DNA composed of TTAGGG (human type) or TTTAGGG (Arabidopsis-type) repeats. Further, we identified TRs across the land plant phylogeny, including common model plants, crop plants, and plants with unusual telomeres. Several lines of functional testing demonstrate the templating telomerase function of the identified TRs and disprove a functionality of the only previously reported plant telomerase RNA in Arabidopsis thaliana. Importantly, our results change the existing paradigm in plant telomere biology which has been based on the existence of a relatively conserved telomerase reverse transcriptase subunit (TERT) associating with highly divergent TRs even between closely related plant taxa. The finding of a monophyletic origin of genuine TRs across land plants opens the possibility to identify TRs directly in transcriptomic or genomic data and/or predict telomere sequences synthesized according to the respective TR template region.

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.

Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure proper chromosome segregation during nuclear divisions and to protect the chromosome ends from deterioration and fusion, respectively. While centromeric sequences may differ between species, with arrays of tandemly repeated sequences and retrotransposons being the most abundant sequence types in plant centromeres, telomeric sequences are usually highly conserved among plants and other organisms. The genome size of the carnivorous genus Genlisea (Lentibulariaceae) is highly variable. Here we study evolutionary sequence plasticity of these chromosomal domains at an intrageneric level. We show that Genlisea nigrocaulis (1C = 86 Mbp; 2n = 40) and G. hispidula (1C = 1550 Mbp; 2n = 40) differ as to their DNA composition at centromeres and telomeres. G. nigrocaulis and its close relative G. pygmaea revealed mainly 161 bp tandem repeats, while G. hispidula and its close relative G. subglabra displayed a combination of four retroelements at centromeric positions. G. nigrocaulis and G. pygmaea chromosome ends are characterized by the Arabidopsis-type telomeric repeats (TTTAGGG); G. hispidula and G. subglabra instead revealed two intermingled sequence variants (TTCAGG and TTTCAGG). These differences in centromeric and, surprisingly, also in telomeric DNA sequences, uncovered between groups with on average a > 9-fold genome size difference, emphasize the fast genome evolution within this genus. Such intrageneric evolutionary alteration of telomeric repeats with cytosine in the guanine-rich strand, not yet known for plants, might impact the epigenetic telomere chromatin modification.

Characterization of histone post-translational modifications (PTMs) is still challenging, and robust histone sample preparation is essential for convincing evaluation of PTMs by mass spectrometry. An effective protocol for extracting plant histone proteins must also avoid excessive co-extraction of the numerous potential interfering compounds, including those related to secondary metabolism. Currently, the co-existence of histone marks is addressed mostly by shotgun proteomic analysis following chemical derivatization of histone lysine residues. Here, we report a straightforward approach for plant histone sample preparation for mass spectrometry, based on filter-aided sample preparation coupled with histone propionylation. The approach offers savings in sample handling and preparation time, enables removal of interfering compounds from the sample, and does not require either precipitation or dialysis of histone extract. We show the comparison of two protocol variants for derivatization of histone proteins, in-solution propionylation in the vial and propionylation on the filter unit. For both protocols, we obtained identical abundances of post-translationally modified histone peptides. Although shorter time is required for histone protein labeling on the filter unit, in-solution derivatization slightly outweighed filter-based variant by lower data variability. Nevertheless, both protocol variants appear to be efficient and convenient approach for preparation of plant histones for mass spectrometric analysis.

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.

Species with holocentric chromosomes are often characterized by a rapid karyotype evolution. In contrast to species with monocentric chromosomes where acentric fragments are lost during cell division, breakage of holocentric chromosomes creates fragments with normal centromere activity. To decipher the mechanism that allows holocentric species an accelerated karyotype evolution via chromosome breakage, we analyzed the chromosome complements of irradiated Luzula elegans plants. The resulting chromosomal fragments and rearranged chromosomes revealed holocentromere-typical CENH3 and histone H2AThr120ph signals as well as the same mitotic mobility like unfragmented chromosomes. Newly synthesized telomeres at break points become detectable 3 weeks after irradiation. The presence of active telomerase suggests a telomerase-based mechanism of chromosome healing. A successful transmission of holocentric chromosome fragments across different generations was found for most offspring of irradiated plants. Hence, a combination of holokinetic centromere activity and the fast formation of new telomeres at break points enables holocentric species a rapid karyotype evolution involving chromosome fissions and rearrangements.

Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are important for the maintenance of genomic stability. Telomeres were considered as typical heterochromatic regions, but in light of recent results, this view should be reconsidered. Asymmetrically located cytosines in plant telomeric DNA repeats may be substrates for a DNA methyltransferase enzyme and indeed, it was shown that these repeats are methylated. Here, we analyse the methylation of telomeric cytosines and the length of telomeres in Arabidopsis thaliana methylation mutants (met 1-3 and ddm 1-8), and in their wild-type siblings that were germinated in the presence of hypomethylation drugs. Our results show that cytosine methylation in telomeric repeats depends on the activity of MET1 and DDM1 enzymes. Significantly shortened telomeres occur in later generations of methylation mutants as well as in plants germinated in the presence of hypomethylation drugs, and this phenotype is stably transmitted to the next plant generation. A possible role of compromised in vivo telomerase action in the observed telomere shortening is hypothesized based on telomere analysis of hypomethylated telomerase knockout plants. Results are discussed in connection with previous data in this field obtained using different model systems.

Dysfunction of chromatin assembly factor 1 in FASCIATA mutants (fas) of Arabidopsis thaliana results in progressive loss of telomeric DNA. Although replicative telomere shortening is typically associated with incomplete resynthesis of their ends by telomerase, no change in telomerase activity could be detected in vitro in extracts from fas mutants. Besides a possible telomerase malfunction, the telomere shortening in fas mutants could presumably be due to problems with conventional replication of telomeres. To distinguish between the possible contribution of suboptimal function of telomerase in fas mutants under in vivo conditions and problems in conventional telomere replication, we crossed fas and tert (telomerase reverse transcriptase) knockout mutants and analyzed telomere shortening in segregated fas mutants, tert mutants, and double fas tert mutants in parallel. We demonstrate that fas tert knockouts show greater replicative telomere shortening than that observed even in the complete absence of telomerase (tert mutants). While the effect of tert and fas mutations on telomere lengths in double mutants is additive, manifestations of telomere dysfunction in double fas tert mutants (frequency of anaphase bridges, onset of chromosome end fusions, and common involvement of 45S rDNA in chromosome fusion sites) are similar to those in tert mutants. We conclude that in addition to possible impairment of telomerase action, a further mechanism contributes to telomere shortening in fas mutants.

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 (∼40%) recovery of GFP-HP1α and GFP-HP1β 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 (∼10% fluorescence recovery) or Polycomb group-related GFP-BMI1 protein (∼20% fluorescence recovery). These findings imply a novel fact that only certain subset of proteins in apoptotic bodies is dynamic.

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.

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.

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.

Telomerase, an enzyme responsible for the maintenance of linear chromosome ends, is precisely regulated during plant development. In animals, involvement of the epigenetic state of the telomerase reverse transcriptase (TERT) gene in the complex regulation of telomerase activity has been reported. To reveal whether epigenetic mechanisms participate in the regulation of plant telomerase, the relationship between telomerase activity in tissues of Arabidopsis thaliana and DNA methylation and histone modifications in the A. thaliana TERT (AtTERT) upstream region was studied. As expected, a gradual decrease of telomerase activity during leaf maturation was observed. A different pattern with a more progressive loss of telomerase activity and AtTERT transcription during leaf development was revealed in MET1 gene-knockout mutants. Analysis of DNA methylation in the AtTERT upstream region showed low levels of methylated cytosines without notable differences between telomerase-positive and telomerase-negative wild-type tissues. Surprisingly, a high level of CG methylation was found in the AtTERT coding region, although this type of methylation is a characteristic attribute of constitutively expressed genes. Analysis of chromatin modifications in the AtTERT upstream region and in exon 5 showed increased loading of the H3K27me3 mark in the telomerase-negative mature leaf compared to telomerase-positive seedlings, whereas H3K4me3, H3K9Ac, and H3K9me2 were approximately at the same level. Consistently, the chromatin structure of the AtTERT gene was maintained. These results are discussed in the context of the general involvement of epigenetic mechanisms in the regulation of gene expression and with respect to similar studies performed in animal models.

Recently, we reported (Majerova et al. 2011) that hypomethylating drugs efficiently decrease methylation of telomeric DNA and activate telomerase without affecting telomere lengths in tobacco. The key issue of the Letter to Editor written by Vaquero-Sedas and Vega-Palas (2011) is, whether our results demonstrating methylation and druginduced hypomethylation of tobacco telomeric DNA repeats do correspond to the status of terminal telomeric sequences, or rather to internal telomeric sequences (ITSs) which might exist in tobacco genome as it is the case in Arabidopsis thaliana, the model organism which Vaquero- Sedas and Vega-Palas have been working with. They are referring to their recent article (Vaquero-Sedas et al. 2010) in which they conclude that ITSs in A. thaliana are heterochromatic, while telomeres are euchromatic. We are pleased that our paper has initiated this debate and take the opportunity to go through the key points of the Letter of Vaquero-Sedas and Vega-Palas

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.

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.

The organisation of dinoflagellate chromosomes is exceptional among eukaryotes. Their genomes are the largest in the Eukarya domain, chromosomes lack histones and may exist in liquid crystalline state. Therefore, the study of the structural and functional properties of dinoflagellate chromosomes is of high interest. In this work, we have analysed the telomeres and telomerase in two Dinoflagellata species, Karenia papilionacea and Crypthecodinium cohnii. Active telomerase, synthesising exclusively Arabidopsis-type telomere sequences, was detected in cell extracts. The terminal position of TTTAGGG repeats was determined by in situ hybridisation and BAL31 digestion methods and provides evidence for the linear characteristic of dinoflagellate chromosomes. The length of telomeric tracts, 25-80 kb, is the largest among unicellular eukaryotic organisms to date. Both the presence of long arrays of perfect telomeric repeats at the ends of dinoflagellate chromosomes and the existence of active telomerase as the primary tool for their high-fidelity maintenance demonstrate the general importance of these structures throughout eukaryotes. We conclude that whilst chromosomes of dinoflagellates are unique in many aspects of their structure and composition, their telomere maintenance follows the most common scenario.

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.

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.

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.

The characterization of unusual telomere sequence sheds light on patterns of telomere evolution, maintenance and function. Plant species from the closely related genera Cestrum, Vestia and Sessea (family Solanaceae) lack known plant telomeric sequences. Here we characterize the telomere of Cestrum elegans, work that was a challenge because of its large genome size and few chromosomes (1C 9.76 pg; n = 8). We developed an approach that combines BAL31 digestion, which digests DNA from the ends and chromosome breaks, with next-generation sequencing (NGS), to generate data analysed in RepeatExplorer, designed for de novo repeats identification and quantification. We identify an unique repeat motif (TTTTTTAGGG)n in C. elegans, occurring in ca. 30 400 copies per haploid genome, averaging ca. 1900 copies per telomere, and synthesized by telomerase. We demonstrate that the motif is synthesized by telomerase. The occurrence of an unusual eukaryote (TTTTTTAGGG)n telomeric motif in C. elegans represents a switch in motif from the 'typical' angiosperm telomere (TTTAGGG)n . That switch may have happened with the divergence of Cestrum, Sessea and Vestia. The shift in motif when it arose would have had profound effects on telomere activity. Thus our finding provides a unique handle to study how telomerase and telomeres responded to genetic change, studies that will shed more light on telomere function.

A comparative approach in biology is needed to assess the universality of rules governing this discipline. In plant telomere research, most of the key principles were established based on studies in only single model plant, Arabidopsis thaliana. These principles include the absence of telomere shortening during plant development and the corresponding activity of telomerase in dividing (meristem) plant cells. Here we examine these principles in Physcomitrella patens as a representative of lower plants. To follow telomerase expression, we first characterize the gene coding for the telomerase reverse transcriptase subunit PpTERT in P. patens, for which only incomplete prediction has been available so far. In protonema cultures of P. patens, growing by filament apical cell division, the proportion of apical (dividing) cells was quantified and telomere length, telomerase expression and activity were determined. Our results show telomere stability and demonstrate proportionality of telomerase activity and expression with the number of apical cells. In addition, we analyze telomere maintenance in mre11, rad50, nbs1, ku70 and lig4 mutants of P. patens and compare the impact of these mutations in double-strand-break (DSB) repair pathways with earlier observations in corresponding A. thaliana mutants. Telomere phenotypes are absent and DSB repair kinetics is not affected in P. patens mutants for DSB factors involved in non-homologous end joining (NHEJ). This is compliant with the overall dominance of homologous recombination over NHEJ pathways in the moss, contrary to the inverse situation in flowering plants.

Chromosome ends - telomeres - are a focus of intensive research due to their importance for the maintenance of chromosome stability. Their shortening du e to incomplete replication functions as a molecular clock counting the number of cell divi sions, and ultimately results in cell-cycle arrest and cellular senescence. Determination of te lomere lengths is an essential approach in telomere biology for research and diagnostic applications. Term inal Restriction Fragments (TRF) analysis is the oldest approach to analyze telomere lengths and remains the “gold standard” even in current studies. This technique relies on the fact that repeated minisatellite telomeric units do not contain target sites for restri ction enzymes. Consequently, telomeres remain in relatively long fragments (TRF), whereas the genomic DNA is digested into short pieces. Fragments of telomeric DNA are then visualized by hybridization with radioactively labeled telomeric probe. As TRF include besides telomeres also a short region of telomere-associated DNA up to the first restriction site, resul ts are slightly shifted towards higher TRFs values. Therefore, the use of frequent cutters or their mixtures is recommended to minimize this difference. Moreover, by using TRF analysis it is possible to distinguish genuine (terminal) telomeres from interstitial telomeric repeats (ITR) (Richa rds and Ausubel, 1988). In this approach, BAL31 digestion is first applied on high molec ular weight DNA. The enzyme progressively degrades linear DNA from its ends. The degrad ed DNA is then digested with one or more restriction enzymes and fragments are separated by gel electrophoresis. After blotting, membranes are probed with either a terminal marker sequence or telomeric sequence. Genuine TRF can be distinguished from ITR due to their progres sive shortening with increasing BAL31 digestion time, while ITR are BAL31-resistan t. The TRF BAL31 digestion pattern at the time zero indicates the approximate telomere lengths (Fajkus et al. , 2005).

Cadmium belongs to the most dangerous environmental pollutants among the toxic heavy metals seriously affecting vital functions in both animal and plant cells. It has been previously shown that cadmium ions at 50–100 μM concentrations caused tobacco BY-2 (TBY-2) cells to enter apoptosis within several days of exposure. Phytochelatins (PCs), the “plant metallothioneins”, are cysteine-rich peptides involved in detoxification of heavy metals in plants. The PCs are synthesized in response to the heavy metal exposure. In this paper, we utilized electrochemical analysis to monitor accumulation of PCs in the TBY-2 cells exposed to cadmium ions. Measurements of a characteristic PC signal at mercury electrode in the presence of cobalt ions made it possible to detect changes in the cellular PC levels during the time of cultivation, starting from 30 min after exposure. Upon TBY-2 cultivation in the presence of cytotoxic cadmium concentrations, the PC levels remarkably increased during the pre-apoptotic phase and reached a limiting value at cultivation times coinciding with apoptosis trigger. The PC level observed for a sub-cytotoxic cadmium concentration (10 μM) was about three-times lower than that observed for the 50 or 100 μM cadmium ions after 5 days of exposure. We show that using a simple electrochemical analysis, synthesis of PCs in plant cells can be easily followed in parallel with other tests of the cellular response to the toxic heavy metal stress.