Telomeres are essential nucleoprotein structures at the very ends of linear eukaryote chromosomes. They shelter the terminal genome territories against degradation and prevent the natural chromosome ends from being recognized by repair mechanisms as double-strand DNA breaks.There are two basic characteristics of telomeric DNA, its sequence and its length. The telomere sequence is important as a "landing area" for specific telomere-binding proteins, which function as signals and moderate the interactions required for correct telomere function. While the sequence forms the proper "landing surface" of telomeric DNA, its length is similarly important. Too short or exceptionally long telomere DNA cannot perform its function properly. In this chapter, methods for the investigation of these two basic telomere DNA characteristics are described, namely, telomere motif identification and telomere length measurement.

• Keywords
• BAL31-NGS, Pulsed-field gel electrophoresis (PFGE), Tandem repeats finder, Telomere, Terminal restriction fragments,
• MeSH
• DNA * genetics   MeSH
• DNA Breaks, Double-Stranded MeSH
• Telomere-Binding Proteins genetics   MeSH
• Telomere * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA * MeSH
• Telomere-Binding Proteins MeSH

Telomeres are essential structures formed from satellite DNA repeats at the ends of chromosomes in most eukaryotes. Satellite DNA repeat sequences are useful markers for karyotyping, but have a more enigmatic role in the eukaryotic cell. Much work has been done to investigate the structure and arrangement of repetitive DNA elements in classical models with implications for species evolution. Still more is needed until there is a complete picture of the biological function of DNA satellite sequences, particularly when considering non-model organisms. Celebrating Gregor Mendel's anniversary by going to the roots, this review is designed to inspire and aid new research into telomeres and satellites with a particular focus on non-model organisms and accessible experimental and in silico methods that do not require specialized equipment or expensive materials. We describe how to identify telomere (and satellite) repeats giving many examples of published (and some unpublished) data from these techniques to illustrate the principles behind the experiments. We also present advice on how to perform and analyse such experiments, including details of common pitfalls. Our examples are a selection of recent developments and underexplored areas of research from the past. As a nod to Mendel's early work, we use many examples from plants and insects, especially as much recent work has expanded beyond the human and yeast models traditional in telomere research. We give a general introduction to the accepted knowledge of telomere and satellite systems and include references to specialized reviews for the interested reader.

• Keywords
• FISH, NGS, TRAP, eukaryotic tree of life, interstitial telomere sequences, retroelements, satellite, subtelomere structure, telomerase RNA, telomere evolution,
• MeSH
• DNA MeSH
• Humans MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• DNA, Satellite * MeSH
• Base Sequence MeSH
• Telomere * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• DNA MeSH
• DNA, Satellite * 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

Non-random gene organization in eukaryotes plays a significant role in genome evolution. Here, we investigate the origin of a biosynthetic gene cluster for production of defence compounds in oat-the avenacin cluster. We elucidate the structure and organisation of this 12-gene cluster, characterise the last two missing pathway steps, and reconstitute the entire pathway in tobacco by transient expression. We show that the cluster has formed de novo since the divergence of oats in a subtelomeric region of the genome that lacks homology with other grasses, and that gene order is approximately colinear with the biosynthetic pathway. We speculate that the positioning of the late pathway genes furthest away from the telomere may mitigate against a 'self-poisoning' scenario in which toxic intermediates accumulate as a result of telomeric gene deletions. Our investigations reveal a striking example of adaptive evolution underpinned by remarkable genome plasticity.

• MeSH
• In Situ Hybridization, Fluorescence MeSH
• Edible Grain genetics   MeSH
• Metabolic Networks and Pathways genetics   MeSH
• Evolution, Molecular MeSH
• Multigene Family MeSH
• Disease Resistance genetics   MeSH
• Avena genetics   metabolism   MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Saponins biosynthesis   chemistry   genetics   MeSH
• Whole Genome Sequencing MeSH
• RNA-Seq MeSH
• Synteny genetics   MeSH
• Nicotiana metabolism   MeSH
• Telomere genetics   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• avenacin A 1 MeSH Browser
• Saponins 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

Understanding how the packaging of chromatin in the nucleus is regulated and organized to guide complex cellular and developmental programmes, as well as responses to environmental cues is a major question in biology. Technological advances have allowed remarkable progress within this field over the last years. However, we still know very little about how the 3D genome organization within the cell nucleus contributes to the regulation of gene expression. The nuclear space is compartmentalized in several domains such as the nucleolus, chromocentres, telomeres, protein bodies, and the nuclear periphery without the presence of a membrane around these domains. The role of these domains and their possible impact on nuclear activities is currently under intense investigation. In this review, we discuss new data from research in plants that clarify functional links between the organization of different nuclear domains and plant genome function with an emphasis on the potential of this organization for gene regulation.

• Keywords
• 3D Chromatin organization, chromocentres, gene expression, liquid–liquid phase separation (LLPS), nuclear bodies, nuclear domains, nuclear periphery, nucleolus, telomeres, topologically associated domains (TADs),
• MeSH
• Cell Nucleolus MeSH
• Cell Nucleus * MeSH
• Chromatin * MeSH
• Gene Expression Regulation MeSH
• Plants genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Chromatin * MeSH

Arabidopsis and human ARM protein interact with telomerase. Deregulated mRNA levels of DNA repair and ribosomal protein genes in an Arabidopsis arm mutant suggest non-telomeric ARM function. The human homolog ARMC6 interacts with hTRF2. Telomerase maintains telomeres and has proposed non-telomeric functions. We previously identified interaction of the C-terminal domain of Arabidopsis telomerase reverse transcriptase (AtTERT) with an armadillo/β-catenin-like repeat (ARM) containing protein. Here we explore protein-protein interactions of the ARM protein, AtTERT domains, POT1a, TRF-like family and SMH family proteins, and the chromatin remodeling protein CHR19 using bimolecular fluorescence complementation (BiFC), yeast two-hybrid (Y2H) analysis, and co-immunoprecipitation. The ARM protein interacts with both the N- and C-terminal domains of AtTERT in different cellular compartments. ARM interacts with CHR19 and TRF-like I family proteins that also bind AtTERT directly or through interaction with POT1a. The putative human ARM homolog co-precipitates telomerase activity and interacts with hTRF2 protein in vitro. Analysis of Arabidopsis arm mutants shows no obvious changes in telomere length or telomerase activity, suggesting that ARM is not essential for telomere maintenance. The observed interactions with telomerase and Myb-like domain proteins (TRF-like family I) may therefore reflect possible non-telomeric functions. Transcript levels of several DNA repair and ribosomal genes are affected in arm mutants, and ARM, likely in association with other proteins, suppressed expression of XRCC3 and RPSAA promoter constructs in luciferase reporter assays. In conclusion, ARM can participate in non-telomeric functions of telomerase, and can also perform its own telomerase-independent functions.

• Keywords
• ARMC6, Armadillo/β-catenin-like repeat, AtTERT, Homologous recombination, Protein–protein interaction, Telomerase activity,
• MeSH
• Arabidopsis enzymology   genetics   MeSH
• Holoenzymes MeSH
• Humans MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Armadillo Domain Proteins genetics   metabolism   MeSH
• Genes, Reporter MeSH
• Two-Hybrid System Techniques MeSH
• Telomerase genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• ARMC6 protein, human MeSH Browser
• Holoenzymes MeSH
• Arabidopsis Proteins MeSH
• Armadillo Domain Proteins MeSH
• Telomerase MeSH

The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.

• Keywords
• AtPOT1a, PURα1, Pontin, Reptin, TAP-MS, Telomerase,
• MeSH
• Arabidopsis enzymology   genetics   MeSH
• Cell Nucleus enzymology   MeSH
• Chromatography, Affinity MeSH
• Gene Expression MeSH
• Protein Interaction Domains and Motifs MeSH
• Cells, Cultured MeSH
• Protein Interaction Mapping MeSH
• Protein Interaction Maps MeSH
• Protein Multimerization MeSH
• Arabidopsis Proteins genetics   isolation & purification   metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Telomerase genetics   isolation & purification   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arabidopsis Proteins MeSH
• Telomerase 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

The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.

• Keywords
• heterochromatin, nuclear architecture, nucleolus, nucleolus-associated chromatin domains, telomere,
• MeSH
• Arabidopsis MeSH
• Cell Nucleolus metabolism   MeSH
• Gene Expression * MeSH
• Phosphoproteins metabolism   MeSH
• Transcription, Genetic genetics   MeSH
• Genome, Plant * MeSH
• Heterochromatin genetics   metabolism   MeSH
• Nucleolin MeSH
• RNA-Binding Proteins metabolism   MeSH
• DNA, Ribosomal genetics   MeSH
• RNA, Ribosomal metabolism   MeSH
• Telomere metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phosphoproteins MeSH
• Heterochromatin MeSH
• RNA-Binding Proteins MeSH
• DNA, Ribosomal MeSH
• RNA, Ribosomal MeSH