The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.

• MeSH
• Arabidopsis embryologie   genetika   imunologie   MeSH
• duplikace genu MeSH
• genom rostlinný MeSH
• oprava DNA * genetika   MeSH
• podjednotky proteinů metabolismus   MeSH
• poškození DNA * genetika   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• pyl genetika   MeSH
• regulace genové exprese u rostlin MeSH
• semena rostlinná genetika   metabolismus   MeSH
• transkriptom genetika   MeSH
• upregulace genetika   MeSH
• vajíčko rostlin genetika   MeSH
• vazba proteinů MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Restoration of transcriptionally silenced genes by means of methyltransferases inhibitors plays a crucial role in the current therapy of myelodysplastic syndromes and certain types of leukemias. A comparative study of hypomethylating activities of a series of 5-azacytidine nucleosides: 5-azacytidine (AC), 2'-deoxy-5-azacytidine (DAC) and its α-anomer (α-DAC), 5,6-dihydro-5-azacytidine (DHAC), 2'-deoxy-5,6-dihydro-5-azacytidine (DHDAC, KP-1212) and its α-anomer (α-DHDAC), and of a 2-pyrimidone ribonucleoside (zebularine) was conducted. Methylation-specific PCR was employed to detect the efficiency of individual agents on cyclin-dependent kinase inhibitor 2B and thrombospondin-1 hypermethylated gene loci. Overall changes in DNA methylation level were quantified by direct estimation of 5-methyl-2'-deoxycytidine-5'-monophosphate by HPLC using digested genomic DNA. Flow cytometric analysis of cell cycle progression and apoptotic markers was used to determine cytotoxicity of the compounds. mRNA expression was measured using qRT-PCR. 2'-deoxy-5,6-dihydro-5-azacytidine was found to be less cytotoxic and more stable than 2'-deoxy-5-azacytidine at the doses that induce comparable DNA hypomethylation and gene reactivation. This makes it a valuable tool for epigenetic research and worth further investigations to elucidate its possible therapeutic potential.

• MeSH
• apoptóza účinky léků   MeSH
• azacytidin analogy a deriváty   chemie   farmakologie   MeSH
• genetické lokusy MeSH
• genom lidský MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• metylace DNA účinky léků   MeSH
• molekulární struktura MeSH
• regulace genové exprese MeSH
• thrombospondin 1 genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Epigenetic variation has been proposed to contribute to the success of asexual plants, either as a contributor to phenotypic plasticity or by enabling transient adaptation via selection on transgenerationally stable, but reversible, epialleles. While recent studies in experimental plant populations have shown the potential for epigenetic mechanisms to contribute to adaptive phenotypes, it remains unknown whether heritable variation in ecologically relevant traits is at least partially epigenetically determined in natural populations. Here, we tested the hypothesis that DNA methylation variation contributes to heritable differences in flowering time within a single widespread apomictic clonal lineage of the common dandelion (Taraxacum officinale s. lat.). Apomictic clone members of the same apomictic lineage collected from different field sites showed heritable differences in flowering time, which was correlated with inherited differences in methylation-sensitive AFLP marker profiles. Differences in flowering between apomictic clone members were significantly reduced after in vivo demethylation using the DNA methyltransferase inhibitor zebularine. This synchronization of flowering times suggests that flowering time divergence within an apomictic lineage was mediated by differences in DNA methylation. While the underlying basis of the methylation polymorphism at functional flowering time-affecting loci remains to be demonstrated, our study shows that epigenetic variation contributes to heritable phenotypic divergence in ecologically relevant traits in natural plant populations. This result also suggests that epigenetic mechanisms can facilitate adaptive divergence within genetically uniform asexual lineages.

• MeSH
• analýza polymorfismu délky amplifikovaných restrikčních fragmentů MeSH
• epigeneze genetická * MeSH
• květy fyziologie   MeSH
• metylace DNA * MeSH
• mikrosatelitní repetice MeSH
• nepohlavní rozmnožování MeSH
• populační genetika MeSH
• Taraxacum genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Finsko MeSH
• Německo MeSH

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.

• MeSH
• Arabidopsis účinky léků   genetika   metabolismus   MeSH
• chromatin genetika   MeSH
• cytidin analogy a deriváty   farmakologie   MeSH
• druhová specificita MeSH
• ekotyp MeSH
• epigeneze genetická účinky léků   MeSH
• histony metabolismus   MeSH
• homeostáza telomer * účinky léků   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mutace genetika   MeSH
• proteiny huseníčku metabolismus   MeSH
• regenerace účinky léků   MeSH
• rostlinné geny MeSH
• tabák genetika   MeSH
• techniky tkáňových kultur * MeSH
• telomerasa metabolismus   MeSH
• telomery metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

DNA methylation inhibitors are being extensively studied as potential anticancer agents. In the present study, we compared the capability of alpha anomer of 5-aza-2'-deoxycytidine (alpha-5-azadCyd) to induce down-regulation of hTERT expression in HL-60 cells with other nucleoside analogs that act as DNA methylation inhibitors: beta-5-azadCyd (decitabine), (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA], isobutyl ester of (R,S)-3-(adenin-9-yl)-2-hydroxypropanoic acid [(R,S)-AHPA-ibu] and prospective DNA methylation inhibitors (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine [(S)-HPMPazaC] and 5-fluoro-zebularine (F-PymRf). Exposure to alpha-5-azadCyd induced the down-regulation of hTERT expression in low micromolar concentrations (0.05-50 microM). A more cytotoxic beta anomer caused a transient up-regulation of hTERT and a subsequent reduction in hTERT mRNA levels at concentrations more than 10 times below its GIC50 value. In this respect, (S)-DHPA and (R,S)-AHPA-ibu were less efficient, since a similar effect was achieved at concentrations above their GIC(50). In contrast, F-PymRf treatment resulted in up to a three-fold induction of hTERT expression within a broad range of concentrations. In all cases, the down-regulation of hTERT expression was concentration-dependent. The correlation was found between c-myc overexpression and transiently elevated hTERT expression after treatment with all tested compounds except for alpha-5-azadCyd and (S)-HPMPazaC. Although the primary task of hypomethylating agents in anticancer therapy lies in reactivation of silenced tumour-suppressor genes, the inhibition of hTERT expression might also be a fruitful clinical effect of this approach.

• MeSH
• antimetabolity antitumorózní farmakologie   MeSH
• azacytidin analogy a deriváty   farmakologie   chemie   MeSH
• DNA metabolismus   MeSH
• down regulace MeSH
• financování organizované MeSH
• HL-60 buňky MeSH
• lidé MeSH
• messenger RNA biosyntéza   MeSH
• metylace DNA účinky léků   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• S-adenosylhomocystein metabolismus   MeSH
• S-adenosylmethionin metabolismus   MeSH
• stereoizomerie MeSH
• telomerasa biosyntéza   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH