Nejvíce citovaný článek - PubMed ID 27377564
Phenotypic reversion in fas mutants of Arabidopsis thaliana by reintroduction of FAS genes: variable recovery of telomeres with major spatial rearrangements and transcriptional reprogramming of 45S rDNA genes
Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.
The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.
- MeSH
- Arabidopsis genetika fyziologie MeSH
- ATM protein genetika metabolismus MeSH
- fyziologický stres MeSH
- genom rostlinný MeSH
- nestabilita genomu MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- protoonkogenní proteiny c-myb genetika metabolismus MeSH
- replikace DNA * MeSH
- signální transdukce * MeSH
- transkripční faktory genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ATM protein, Arabidopsis MeSH Prohlížeč
- ATM protein MeSH
- ATR1 protein, Arabidopsis MeSH Prohlížeč
- FAS protein, Arabidopsis MeSH Prohlížeč
- protein-serin-threoninkinasy MeSH
- proteiny huseníčku MeSH
- protoonkogenní proteiny c-myb MeSH
- SOG1 protein, Arabidopsis MeSH Prohlížeč
- transkripční faktory MeSH
- WEE1 protein, Arabidopsis MeSH Prohlížeč
Genes encoding ribosomal RNA (rDNA) are essential for cell survival and are particularly sensitive to factors leading to genomic instability. Their repetitive character makes them prone to inappropriate recombinational events arising from collision of transcriptional and replication machineries, resulting in unstable rDNA copy numbers. In this review, we summarize current knowledge on the structure and organization of rDNA, its role in sensing changes in the genome, and its linkage to aging. We also review recent findings on the main factors involved in chromatin assembly and DNA repair in the maintenance of rDNA stability in the model plants Arabidopsis thaliana and the moss Physcomitrella patens, providing a view across the plant evolutionary tree.
- Klíčová slova
- CAF-1, RAD51, RTEL1, genome stability, rDNA organization, rRNA genes, ribosome,
- MeSH
- Arabidopsis genetika MeSH
- DNA rostlinná genetika MeSH
- genetická transkripce MeSH
- genová dávka MeSH
- lidé MeSH
- mechy genetika MeSH
- nestabilita genomu MeSH
- oprava DNA * MeSH
- replikace DNA MeSH
- restrukturace chromatinu MeSH
- ribozomální DNA genetika MeSH
- stárnutí genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- DNA rostlinná MeSH
- ribozomální DNA MeSH
Rapid plant genome evolution is crucial to adapt to environmental changes. Chromosomal rearrangements and gene copy number variation (CNV) are two important tools for genome evolution and sources for the creation of new genes. However, their emergence takes many generations. In this study, we show that in Arabidopsis thaliana, a significant loss of ribosomal RNA (rRNA) genes with a past history of a mutation for the chromatin assembly factor 1 (CAF1) complex causes rapid changes in the genome structure. Using long-read sequencing and microscopic approaches, we have identified up to 15 independent large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. Our data suggest that these TDDOs appeared within a few generations, leading to the duplication of hundreds of genes. By subsequently focusing on a line only containing 20% of rRNA gene copies (20rDNA line), we investigated the impact of TDDOs on 3D genome organization, gene expression, and cytosine methylation. We found that duplicated genes often accumulate more transcripts. Among them, several are involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and discuss their potential implications for the evolution of plant genomes.
Genes encoding 45S ribosomal RNA (rDNA) are known for their abundance within eukaryotic genomes and for their unstable copy numbers in response to changes in various genetic and epigenetic factors. Commonly, we understand as epigenetic factors (affecting gene expression without a change in DNA sequence), namely DNA methylation, histone posttranslational modifications, histone variants, RNA interference, nucleosome remodeling and assembly, and chromosome position effect. All these were actually shown to affect activity and stability of rDNA. Here, we focus on another phenomenon - the potential of DNA containing shortly spaced oligo-guanine tracts to form quadruplex structures (G4). Interestingly, sites with a high propensity to form G4 were described in yeast, animal, and plant rDNAs, in addition to G4 at telomeres, some gene promoters, and transposons, suggesting the evolutionary ancient origin of G4 as a regulatory module. Here, we present examples of rDNA promoter regions with extremely high potential to form G4 in two model plants, Arabidopsis thaliana and Physcomitrella patens. The high G4 potential is balanced by the activity of G4-resolving enzymes. The ability of rDNA to undergo these "structural gymnastics" thus represents another layer of the rich repertoire of epigenetic regulations, which is pronounced in rDNA due to its highly repetitive character.
- Klíčová slova
- G4, quadruplex DNA, rDNA stability, replication, ribosomal RNA genes, transcription,
- Publikační typ
- časopisecké články MeSH
Approximately seven hundred 45S rRNA genes (rDNA) in the Arabidopsis thaliana genome are organised in two 4 Mbp-long arrays of tandem repeats arranged in head-to-tail fashion separated by an intergenic spacer (IGS). These arrays make up 5 % of the A. thaliana genome. IGS are rapidly evolving sequences and frequent rearrangements inside the rDNA loci have generated considerable interspecific and even intra-individual variability which allows to distinguish among otherwise highly conserved rRNA genes. The IGS has not been comprehensively described despite its potential importance in regulation of rDNA transcription and replication. Here we describe the detailed sequence variation in the complete IGS of A. thaliana WT plants and provide the reference/consensus IGS sequence, as well as genomic DNA analysis. We further investigate mutants dysfunctional in chromatin assembly factor-1 (CAF-1) (fas1 and fas2 mutants), which are known to have a reduced number of rDNA copies, and plant lines with restored CAF-1 function (segregated from a fas1xfas2 genetic background) showing major rDNA rearrangements. The systematic rDNA loss in CAF-1 mutants leads to the decreased variability of the IGS and to the occurrence of distinct IGS variants. We present for the first time a comprehensive and representative set of complete IGS sequences, obtained by conventional cloning and by Pacific Biosciences sequencing. Our data expands the knowledge of the A. thaliana IGS sequence arrangement and variability, which has not been available in full and in detail until now. This is also the first study combining IGS sequencing data with RFLP analysis of genomic DNA.
- Klíčová slova
- 45S ribosomal DNA, Arabidopsis thaliana, Chromatin assembly factor, Intergenic spacer, Nucleolus organizer region, rDNA rearrangements,
- MeSH
- Arabidopsis genetika MeSH
- faktor 1 pro uspořádání chromatinu genetika MeSH
- genetická variace genetika MeSH
- mezerníky ribozomální DNA genetika MeSH
- mutace MeSH
- repetitivní sekvence nukleových kyselin genetika MeSH
- RNA ribozomální genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- faktor 1 pro uspořádání chromatinu MeSH
- mezerníky ribozomální DNA MeSH
- RNA ribozomální MeSH
- RNA, ribosomal, 45S MeSH Prohlížeč
