SMC5/6 complex-mediated SUMOylation stimulates DNA-protein cross-link repair in Arabidopsis.

DNA-protein cross-links (DPCs) are highly toxic DNA lesions consisting of proteins covalently attached to chromosomal DNA. Unrepaired DPCs physically block DNA replication and transcription. Three DPC repair pathways have been identified in Arabidopsis (Arabidopsis thaliana) to date: the endonucleolytic cleavage of DNA by the structure-specific endonuclease MUS81; proteolytic degradation of the crosslinked protein by the metalloprotease WSS1A; and cleavage of the cross-link phosphodiester bonds by the tyrosyl phosphodiesterases TDP1 and TDP2. Here we describe the evolutionary conserved STRUCTURAL MAINTENANCE OF CHROMOSOMEs SMC5/6 complex as a crucial component involved in DPC repair. We identified multiple alleles of the SMC5/6 complex core subunit gene SMC6B via a forward-directed genetic screen designed to identify the factors involved in the repair of DPCs induced by the cytidine analog zebularine. We monitored plant growth and cell death in response to DPC-inducing chemicals, which revealed that the SMC5/6 complex is essential for the repair of several types of DPCs. Genetic interaction and sensitivity assays showed that the SMC5/6 complex works in parallel to the endonucleolytic and proteolytic pathways. The repair of zebularine-induced DPCs was associated with SMC5/6-dependent SUMOylation of the damage sites. Thus, we present the SMC5/6 complex as an important factor in plant DPC repair.

Popular Gym Fitness Sport: An Analysis of 1387 Recreational Athletes Regarding Prone to Pain Exercises and the Corresponding Localisations.

BACKGROUND: Recreational fitness sports are popular worldwide and rank first among organised sports. This study aims to bridge a knowledge gap by examining which exercises are most prone to causing pain symptoms, as a possible precursor for injury, and analysing the body regions that are most frequently affected. METHODS: Using an online questionnaire, 20 demographic and training-specific items and 49 sport-specific exercises were recorded. Frequent exercises as well as the incidence and distribution of pain symptoms that the athletes experienced during or in relation to their training were evaluated. RESULTS: The study assessed common exercises and documented the frequency and distribution of pain symptoms experienced by athletes during or in relation to their training. A total of 1387 respondents were included in this study. Of these, 732 (53.1%) experienced pain during their fitness training, with 333 (24.2%) being female and 397 (22.3%) being male. The method of creating a training plan showed a significant influence (p < 0.001): athletes who devised their own plans reported pain or instability more frequently than those in the comparison groups. Guided exercises on machines resulted in the lowest frequency of pain (11.54%), while exercises with free weights were associated with the highest pain rate among respondents (19.94%). Specifically, exercises such as the back squat, deadlift, bench press, and triceps dips were identified as the exercises most commonly associated with pain. The most frequently reported pain region was the shoulder, followed by the lower back and knees. CONCLUSION: The findings reveal a significant number of unreported pain symptoms. The disparity between rigorous training volumes and the absence of professional care frequently leads to injuries and pain. It is incumbent upon sports medicine to investigate the root causes of these complaints (pain or instability) to implement preventive measures against potential injuries.

Zebularine induces enzymatic DNA-protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei.

Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA-protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.

Comparative analysis of epigenetic inhibitors reveals different degrees of interference with transcriptional gene silencing and induction of DNA damage.

Repetitive DNA sequences and some genes are epigenetically repressed by transcriptional gene silencing (TGS). When genetic mutants are not available or problematic to use, TGS can be suppressed by chemical inhibitors. However, informed use of epigenetic inhibitors is partially hampered by the absence of any systematic comparison. In addition, there is emerging evidence that epigenetic inhibitors cause genomic instability, but the nature of this damage and its repair remain unclear. To bridge these gaps, we compared the effects of 5-azacytidine (AC), 2'-deoxy-5-azacytidine (DAC), zebularine and 3-deazaneplanocin A (DZNep) on TGS and DNA damage repair. The most effective inhibitor of TGS was DAC, followed by DZNep, zebularine and AC. We confirmed that all inhibitors induce DNA damage and suggest that this damage is repaired by multiple pathways with a critical role of homologous recombination and of the SMC5/6 complex. A strong positive link between the degree of cytidine analog-induced DNA demethylation and the amount of DNA damage suggests that DNA damage is an integral part of cytidine analog-induced DNA demethylation. This helps us to understand the function of DNA methylation in plants and opens the possibility of using epigenetic inhibitors in biotechnology.

Genome invasion by a hypomethylated satellite repeat in Australian crucifer Ballantinia antipoda.

Repetitive sequences are ubiquitous components of all eukaryotic genomes. They contribute to genome evolution and the regulation of gene transcription. However, the uncontrolled activity of repetitive sequences can negatively affect genome functions and stability. Therefore, repetitive DNAs are embedded in a highly repressive heterochromatic environment in plant cell nuclei. Here, we analyzed the sequence, composition and the epigenetic makeup of peculiar non-pericentromeric heterochromatic segments in the genome of the Australian crucifer Ballantinia antipoda. By the combination of high throughput sequencing, graph-based clustering and cytogenetics, we found that the heterochromatic segments consist of a mixture of unique sequences and an A-T-rich 174 bp satellite repeat (BaSAT1). BaSAT1 occupies about 10% of the B. antipoda nuclear genome in >250 000 copies. Unlike many other highly repetitive sequences, BaSAT1 repeats are hypomethylated; this contrasts with the normal patterns of DNA methylation in the B. antipoda genome. Detailed analysis of several copies revealed that these non-methylated BaSAT1 repeats were also devoid of heterochromatic histone H3K9me2 methylation. However, the factors decisive for the methylation status of BaSAT1 repeats remain currently unknown. In summary, we show that even highly repetitive sequences can exist as hypomethylated in the plant nuclear genome.

Analysis of DNA Methylation Content and Patterns in Plants.

DNA methylation is an epigenetic modification, which contributes to the regulation of gene expression and chromatin organization, and thus plays a role in many aspects of plant life. Here we present three methods for the detection of DNA methylation in plant tissues: high performance liquid chromatography, methylation-sensitive restriction digest followed by quantitative PCR and bisulfite conversion followed by single read sequencing. These methods are complementary and allow analysis of DNA methylation in samples from both model and non-model plant species.

Six-Rowed Spike3 (VRS3) Is a Histone Demethylase That Controls Lateral Spikelet Development in Barley.

The complex nature of crop genomes has long prohibited the efficient isolation of agronomically relevant genes. However, recent advances in next-generation sequencing technologies provide new ways to accelerate fine-mapping and gene isolation in crops. We used RNA sequencing of allelic six-rowed spike3 (vrs3) mutants with altered spikelet development for gene identification and functional analysis in barley (Hordeum vulgare). Variant calling in two allelic vrs3 mutants revealed that VRS3 encodes a putative histone Lys demethylase with a conserved zinc finger and Jumonji C and N domain. Sanger sequencing of this candidate gene in independent allelic vrs3 mutants revealed a series of mutations in conserved domains, thus confirming our candidate as the VRS3 gene and suggesting that the row type in barley is determined epigenetically. Global transcriptional profiling in developing shoot apical meristems of vrs3 suggested that VRS3 acts as a transcriptional activator of the row-type genes VRS1 (Hv.HOMEOBOX1) and INTERMEDIUM-C (INT-C; Hv.TEOSINTE BRANCHED1). Comparative transcriptome analysis of the row-type mutants vrs3, vrs4 (Hv.RAMOSA2), and int-c confirmed that all three genes act as transcriptional activators of VRS1 and quantitative variation in the expression levels of VRS1 in these mutants correlated with differences in the number of developed lateral spikelets. The identification of genes and pathways affecting seed number in small grain cereals will enable to further unravel the transcriptional networks controlling this important yield component.

Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis.

DNA damage repair is an essential cellular mechanism that maintains genome stability. Here, we show that the nonmethylable cytidine analog zebularine induces a DNA damage response in Arabidopsis thaliana, independent of changes in DNA methylation. In contrast to genotoxic agents that induce damage in a cell cycle stage-independent manner, zebularine induces damage specifically during strand synthesis in DNA replication. The signaling of this damage is mediated by additive activity of ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED and ATAXIA TELANGIECTASIA MUTATED kinases, which cause postreplicative cell cycle arrest and increased endoreplication. The repair requires a functional STRUCTURAL MAINTENANCE OF CHROMOSOMES5 (SMC5)-SMC6 complex and is accomplished predominantly by synthesis-dependent strand-annealing homologous recombination. Here, we provide insight into the response mechanism for coping with the genotoxic effects of zebularine and identify several components of the zebularine-induced DNA damage repair pathway.

Functional mapping of the plant small RNA methyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins.

Methylation of 3'-terminal nucleotides of miRNA/miRNA* is part of miRNAs biogenesis in plants but is not found in animals. In Arabidopsis thaliana this reaction is carried out by a multidomain AdoMet-dependent 2'-O-methyltransferase HEN1. Using deletion and structure-guided mutational analysis, we show that the double-stranded RNA-binding domains R(1) and R(2) of HEN1 make significant but uneven contributions to substrate RNA binding, and map residues in each domain responsible for this function. Using GST pull-down assays and yeast two-hybrid analysis we demonstrate direct HEN1 interactions, mediated by its FK506-binding protein-like domain and R(2) domain, with the microRNA biogenesis protein HYL1. Furthermore, we find that HEN1 forms a complex with DICER-LIKE 1 (DCL1) ribonuclease, another key protein involved in miRNA biogenesis machinery. In contrast, no direct interaction is detectable between HEN1 and SERRATE. On the basis of these findings, we propose a mechanism of plant miRNA maturation which involves binding of the HEN1 methyltransferase to the DCL1•HYL1•miRNA complex excluding the SERRATE protein.

DNA methylation maintenance consolidates RNA-directed DNA methylation and transcriptional gene silencing over generations in Arabidopsis thaliana.

In plants, 24 nucleotide short interfering RNAs serve as a signal to direct cytosine methylation at homologous DNA regions in the nucleus. If the targeted DNA has promoter function, this RNA-directed DNA methylation may result in transcriptional gene silencing. In a genetic screen for factors involved in RNA-directed transcriptional silencing of a ProNOS-NPTII reporter transgene in Arabidopsis thaliana, we captured alleles of DOMAINS REARRANGED METHYLTRANSFERASE 2, the gene encoding the DNA methyltransferase that is mainly responsible for de novo DNA methylation in the context of RNA-directed DNA methylation. Interestingly, methylation of the reporter gene ProNOS was not completely erased in these mutants, but persisted in the symmetric CG context, indicating that RNA-directed DNA methylation had been consolidated by DNA methylation maintenance. Taking advantage of the segregation of the transgenes giving rise to ProNOS short interfering RNAs and carrying the ProNOS-NPTII reporter in our experimental system, we found that ProNOS DNA methylation maintenance was first evident after two generations of ongoing RNA-directed DNA methylation, and then increased in extent with further generations. As ProNOS DNA methylation had already reached its final level in the first generation of RNA-directed DNA methylation, our findings suggest that establishment of DNA methylation at a particular region may be divided into distinct stages. An initial phase of efficient, but still fully reversible, de novo DNA methylation and transcriptional gene silencing is followed by transition to efficient maintenance of cytosine methylation in a symmetric sequence context accompanied by persistence of gene silencing.

Meristem-specific expression of epigenetic regulators safeguards transposon silencing in Arabidopsis.

In plants, transposable elements (TEs) are kept inactive by transcriptional gene silencing (TGS). TGS is established and perpetuated by RNA-directed DNA methylation (RdDM) and maintenance methylation pathways, respectively. Here, we describe a novel RdDM function specific for shoot apical meristems that reinforces silencing of TEs during early vegetative growth. In meristems, RdDM counteracts drug-induced interference with TGS maintenance and consequently prevents TE activation. Simultaneous disturbance of both TGS pathways leads to transcriptionally active states of repetitive sequences that are inherited by somatic tissues and partially by the progeny. This apical meristem-specific mechanism is mediated by increased levels of TGS factors and provides a checkpoint for correct epigenetic inheritance during the transition from vegetative to reproductive phase and to the next generation.

IDN2 has a role downstream of siRNA formation in RNA-directed DNA methylation.

In plants, a particular class of short interfering (si)RNAs can serve as a signal to induce cytosine methylation at homologous genomic regions. If the targeted DNA has promoter function, this RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing (TGS). RNA-directed transcriptional gene silencing (RdTGS) of transgenes provides a versatile system for the study of epigenetic gene regulation. We used transcription of a nopaline synthase promoter (ProNOS)-inverted repeat (IR) to provide a RNA signal that triggers de novo cytosine methylation and TGS of a homologous ProNOS copy in trans. Utilizing a ProNOS-NPTII reporter gene showing high sensitivity to silencing in this two component system, a forward genetic screen for EMS-induced no rna-directed transcriptional silencing (nrd) mutations was performed in Arabidopsis thaliana. Three nrd mutant lines were found to contain one novel loss-of-function allele of idn2/rdm12 and two of nrpd2a/nrpe2a. IDN2/RDM12 encodes a XH/XS domain protein that is able to bind double-stranded RNA with 5' overhangs, while NRPD2a/NRPE2a encodes the common second-largest subunit of the plant specific DNA-dependent RNA polymerases IV and V involved in silencing processes. Both idn2/rdm12 and nrpd2a/nrpe2a release target transgene expression and reduce CHH context methylation at transgenic as well as endogenous RdDM target regions to similar extents. Nevertheless, accumulation of IR-derived siRNA is not affected, allowing us to present a refined model for the pathway of RdDM and RdTGS that positions function of IDN2 downstream of siRNA formation and points to an important role for its XH domain.

Approved IFCC reference method for the measurement of HbA1c in human blood.

HbA1C is the stable glucose adduct to the N-terminal group of the beta-chain of HbA0. The measurement of HbA1c in human blood is most important for the long-term control of the glycaemic state in diabetic patients. Because there was no internationally agreed reference method the IFCC Working Group on HbA1c Standardization developed a reference method which is here described. In a first step haemoglobin is cleaved into peptides by the enzyme endoproteinase Glu-C, and in a second step the glycated and non-glycated N-terminal hexapeptides of the beta-chain obtained are separated and quantified by HPLC and electrospray ionisation mass spectrometry or in a two-dimensional approach using HPLC and capillary electrophoresis with UV-detection. Both principles give identical results. HbA1c is measured as ratio between the glycated and non-glycated hexapeptides. Calibrators consisting of mixtures of highly purified HbA1c and HbA0 are used. The analytical performance of the reference method has been evaluated by an international network of reference laboratories comprising laboratories from Europe, Japan and the USA. The intercomparison studies of the network showed excellent results with intra-laboratory CVs of 0.5 to 2% and inter-laboratory CVs of 1.4 to 2.3%. Possible interferences have been carefully investigated. Due to the higher specificity of the reference method the results are lower than those generated with most of the present commercial methods which currently are calibrated with unspecific designated comparison methods. The new reference method has been approved by the member societies of the International Federation of Clinical Chemistry and Laboratory Medicine and will be the basis for the future uniform standardization of HbA1c routine assays worldwide.