At the molecular scale, adaptive advantages during plant growth and development rely on modulation of gene expression, primarily provided by epigenetic machinery. One crucial part of this machinery is histone posttranslational modifications, which form a flexible system, driving transient changes in chromatin, and defining particular epigenetic states. Posttranslational modifications work in concert with replication-independent histone variants further adapted for transcriptional regulation and chromatin repair. However, little is known about how such complex regulatory pathways are orchestrated and interconnected in cells. In this work, we demonstrate the utility of mass spectrometry-based approaches to explore how different epigenetic layers interact in Arabidopsis mutants lacking certain histone chaperones. We show that defects in histone chaperone function (e.g., chromatin assembly factor-1 or nucleosome assembly protein 1 mutations) translate into an altered epigenetic landscape, which aids the plant in mitigating internal instability. We observe changes in both the levels and distribution of H2A.W.7, altogether with partial repurposing of H3.3 and changes in the key repressive (H3K27me1/2) or euchromatic marks (H3K36me1/2). These shifts in the epigenetic profile serve as a compensatory mechanism in response to impaired integration of the H3.1 histone in the fas1 mutants. Altogether, our findings suggest that maintaining genome stability involves a two-tiered approach. The first relies on flexible adjustments in histone marks, while the second level requires the assistance of chaperones for histone variant replacement.

• Keywords
• Arabidopsis, chromatin remodeling, histone chaperone complex, histone variants, immunochemistry, mass spectrometry, post-translational modifications,
• MeSH
• Arabidopsis * genetics   metabolism   MeSH
• Epigenesis, Genetic * MeSH
• Chromatin Assembly Factor-1 metabolism   genetics   MeSH
• Histone Chaperones * metabolism   genetics   MeSH
• Histones * metabolism   MeSH
• Mutation MeSH
• Protein Processing, Post-Translational MeSH
• Arabidopsis Proteins * metabolism   genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin Assembly Factor-1 MeSH
• Histone Chaperones * MeSH
• Histones * MeSH
• Arabidopsis Proteins * MeSH

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 genetics   physiology   MeSH
• Ataxia Telangiectasia Mutated Proteins genetics   metabolism   MeSH
• Stress, Physiological MeSH
• Genome, Plant MeSH
• Genomic Instability MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-myb genetics   metabolism   MeSH
• DNA Replication * MeSH
• Signal Transduction * MeSH
• Transcription Factors genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATM protein, Arabidopsis MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• ATR1 protein, Arabidopsis MeSH Browser
• FAS protein, Arabidopsis MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• Arabidopsis Proteins MeSH
• Proto-Oncogene Proteins c-myb MeSH
• SOG1 protein, Arabidopsis MeSH Browser
• Transcription Factors MeSH
• WEE1 protein, Arabidopsis MeSH Browser

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.

• Keywords
• CAF-1, RAD51, RTEL1, genome stability, rDNA organization, rRNA genes, ribosome,
• MeSH
• Arabidopsis genetics   MeSH
• DNA, Plant genetics   MeSH
• Transcription, Genetic MeSH
• Gene Dosage MeSH
• Humans MeSH
• Bryopsida genetics   MeSH
• Genomic Instability MeSH
• DNA Repair * MeSH
• DNA Replication MeSH
• Chromatin Assembly and Disassembly MeSH
• DNA, Ribosomal genetics   MeSH
• Aging genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA, Ribosomal MeSH

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.

• Keywords
• G4, quadruplex DNA, rDNA stability, replication, ribosomal RNA genes, transcription,
• Publication type
• Journal Article 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.

• Keywords
• 45S ribosomal DNA, Arabidopsis thaliana, Chromatin assembly factor, Intergenic spacer, Nucleolus organizer region, rDNA rearrangements,
• MeSH
• Arabidopsis genetics   MeSH
• Chromatin Assembly Factor-1 genetics   MeSH
• Genetic Variation genetics   MeSH
• DNA, Ribosomal Spacer genetics   MeSH
• Mutation MeSH
• Repetitive Sequences, Nucleic Acid genetics   MeSH
• RNA, Ribosomal genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin Assembly Factor-1 MeSH
• DNA, Ribosomal Spacer MeSH
• RNA, Ribosomal MeSH
• RNA, ribosomal, 45S MeSH Browser