Genome stability in the uvh6 mutant of Arabidopsis thaliana.

Plant XPD homolog UVH6 is the protein involved in the repair of strand breaks, and the excision repair and uvh6 mutant is not impaired in transgenerational increase in HRF. While analyzing the transgenerational response to stress in plants, we found that the promoter and gene body of Arabidopsis thaliana (Arabidopsis) XPD homolog UVH6 underwent hypomethylation and showed an increase in the level of transcript. Here, we analyzed the mutant of this gene, uvh6-1, by crossing it to two different reporter lines: one which allows for analysis of homologous recombination frequency (HRF) and another which makes it possible to analyze the frequency of point mutations. We observed that uvh6-1 plants exhibited lower rate of spontaneous homologous recombination but higher frequencies of spontaneous point mutations. The analysis of strand breaks using ROPS and Comet assays showed that the mutant had a much higher level of strand breaks at non-induced conditions. Exposure to stresses such as UVC, heat, cold, flood and drought showed that the mutant was not impaired in an increase in somatic HRF. The analysis of spontaneous HRF in the progeny of control plants compared to that of the progeny of stressed plants demonstrated that uvh6-1 was mildly affected in response to temperature, UV and drought. Our data suggest that UVH6 may be involved in the repair of strand breaks and excision repair, but it is unlikely that UVH6 is required for transgenerational increase in HRF.

Genome stability of Arabidopsis atm, ku80 and rad51b mutants: somatic and transgenerational responses to stress.

DNA double-strand breaks (DSBs) can be repaired via two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). Our previous work showed that exposure to abiotic stresses resulted in an increase in point mutation frequency (PMF) and homologous recombination frequency (HRF), and these changes were heritable. We hypothesized that mutants impaired in DSB recognition and repair would also be deficient in somatic and transgenerational changes in PMF and HRF. To test this hypothesis, we analyzed the genome stability of the Arabidopsis thaliana mutants deficient in ATM (communication between DNA strand break recognition and the repair machinery), KU80 (deficient in NHEJ) and RAD51B (deficient in HR repair) genes. We found that all three mutants exhibited higher levels of DSBs. Plants impaired in ATM had a lower spontaneous PMF and HRF, whereas ku80 plants had higher frequencies. Plants impaired in RAD51B had a lower HRF. HRF in wild-type, atm and rad51b plants increased in response to several abiotic stressors, whereas it did not increase in ku80 plants. The progeny of stressed wild-type and ku80 plants exhibited an increase in HRF in response to all stresses, and the increase was higher in ku80 plants. The progeny of atm plants showed an increase in HRF only when the parental generation was exposed to cold or flood, whereas the progeny of rad51b plants completely lacked a transgenerational increase in HRF. Our experiments showed that mutants impaired in the recognition and repair of DSBs exhibited changes in the efficiency of DNA repair as reflected by changes in strand breaks, point mutation and HRF. They also showed that the HR RAD51B protein and the protein ATM that recognized damaged DNA might play an important role in transgenerational changes in HRF.

Genome-wide Detection of Chimeric Transcripts in Early-stage Non-small Cell Lung Cancer.

BACKGROUND/AIM: Lung cancer remains the main culprit in cancer-related mortality worldwide. Transcript fusions play a critical role in the initiation and progression of multiple cancers. Treatment approaches based on specific targeting of discovered driver events, such as mutations in EGFR, and fusions in NTRK, ROS1, and ALK genes led to profound improvements in clinical outcomes. The formation of chimeric proteins due to genomic rearrangements or at the post-transcriptional level is widespread and plays a critical role in tumor initiation and progression. Yet, the fusion landscape of lung cancer remains underexplored. MATERIALS AND METHODS: We used the JAFFA pipeline to discover transcript fusions in early-stage non-small cell lung cancer (NSCLC). The set of detected fusions was further analyzed to identify recurrent events, genes with multiple partners and fusions with high predicted oncogenic potential. Finally, we used a generalized linear model (GLM) to establish statistical associations between fusion occurrences and clinicopathological variables. RNA sequencing was used to discover and characterize transcript fusions in 270 NSCLC samples selected from the Glans-Look specimen repository. The samples were obtained during the early stages of disease prior to the initiation of chemo- or radiotherapy. RESULTS: We identified a set of 792 fusions where 751 were novel, and 33 were recurrent. Four of the 33 recurrent fusions were significantly associated with clinicopathological variables. Several of the fusion partners were represented by well-established oncogenes ERBB4, BRAF, FGFR2, and MET. CONCLUSION: The data presented in this study allow researchers to identify, select, and validate promising candidates for targeted clinical interventions.

Two-stage tuberculosis diagnostics: combining centrifugal microfluidics to detect TB infection and Inh and Rif resistance at the point of care with subsequent antibiotic resistance profiling by targeted NGS.

Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml-1, a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.

ddm1 plants are sensitive to methyl methane sulfonate and NaCl stresses and are deficient in DNA repair.

UNLABELLED: Plant response to stress includes changes in gene expression and chromatin structure. Our previous work showed that Arabidopsis thaliana Dicer-like (DCL) mutants were impaired in transgenerational response to stress that included an increase in recombination frequency, cytosine methylation and stress tolerance. It can be hypothesized that changes in chromatin structure are important for an efficient stress response. To test this hypothesis, we analyzed the stress response of ddm1, a mutant impaired in DDM1, a member of the SWI/SNF family of adenosine triphosphate-dependent chromatin remodeling genes. We exposed Arabidopsis thaliana ddm1 mutants to methyl methane sulfonate (MMS) and NaCl and found that these plants were more sensitive. At the same time, ddm1 plants were similar to wild-type plants in sensitivity to temperature and bleomycin stresses. Direct comparison to met1 plants, deficient in maintenance methyltransferase MET1, showed higher sensitivity of ddm1 plants to NaCl. The level of DNA strand breaks upon exposure to MMS increased in wild-type plants but decreased in ddm1 plants. DNA methylation analysis showed that heterozygous ddm1/DDM1 plants had lower methylation as compared to fourth generation of homozygous ddm1/ddm1 plants. Exposure to MMS resulted in a decrease in methylation in wild-type plants and an increase in ddm1 plants. Finally, in vitro DNA excision repair assay showed lower capacity for ddm1 mutant. Our results provided a new example of a link between genetic genome stability and epigenetic genome stability. KEY MESSAGE: We demonstrate that heterozygous ddm1/DDM1 plants are more sensitive to stress and have more severe changes in methylation than homozygous ddm1/ddm1 plants.

Microsatellite instability in Arabidopsis increases with plant development.

Plant development consists of the initial phase of intensive cell division followed by continuous genome endoreduplication, cell growth, and elongation. The maintenance of genome stability under these conditions is the main task performed by DNA repair and genome surveillance mechanisms. Our previous work showed that the rate of homologous recombination repair in older plants decreases. We hypothesized that this age-dependent decrease in the recombination rate is paralleled with other changes in DNA repair capacity. Here, we analyzed microsatellite stability using transgenic Arabidopsis (Arabidopsis thaliana) plants that carry the nonfunctional ß-glucuronidase gene disrupted by microsatellite repeats. We found that microsatellite instability increased dramatically with plant age. We analyzed the contribution of various mechanisms to microsatellite instability, including replication errors and mistakes of DNA repair mechanisms such as mismatch repair, excision repair, and strand break repair. Analysis of total DNA polymerase activity using partially purified protein extracts showed an age-dependent decrease in activity and an increase in fidelity. Analysis of the steady-state RNA level of DNA replicative polymerases α, δ, Pol I-like A, and Pol I-like B and the expression of mutS homolog 2 (Msh2) and Msh6 showed an age-dependent decrease. An in vitro repair assay showed lower efficiency of nonhomologous end joining in older plants, paralleled by an increase in Ku70 gene expression. Thus, we assume that the more frequent involvement of nonhomologous end joining in strand break repair and the less efficient end-joining repair together with lower levels of mismatch repair activities may be the main contributors to the observed age-dependent increase in microsatellite instability.

Tobacco mosaic virus infection results in an increase in recombination frequency and resistance to viral, bacterial, and fungal pathogens in the progeny of infected tobacco plants.

Our previous experiments showed that infection of tobacco (Nicotiana tabacum) plants with Tobacco mosaic virus (TMV) leads to an increase in homologous recombination frequency (HRF). The progeny of infected plants also had an increased rate of rearrangements in resistance gene-like loci. Here, we report that tobacco plants infected with TMV exhibited an increase in HRF in two consecutive generations. Analysis of global genome methylation showed the hypermethylated genome in both generations of plants, whereas analysis of methylation via 5-methyl cytosine antibodies demonstrated both hypomethylation and hypermethylation. Analysis of the response of the progeny of infected plants to TMV, Pseudomonas syringae, or Phytophthora nicotianae revealed a significant delay in symptom development. Infection of these plants with TMV or P. syringae showed higher levels of induction of PATHOGENESIS-RELATED GENE1 gene expression and higher levels of callose deposition. Our experiments suggest that viral infection triggers specific changes in progeny that promote higher levels of HRF at the transgene and higher resistance to stress as compared with the progeny of unstressed plants. However, data reported in these studies do not establish evidence of a link between recombination frequency and stress resistance.

Local infection with oilseed rape mosaic virus promotes genetic rearrangements in systemic Arabidopsis tissue.

We have previously shown that local infection of tobacco plants with tobacco mosaic virus (TMV) or oilseed rape mosaic virus (ORMV) results in a systemic increase in the homologous recombination frequency (HRF). Here, we analyzed what other changes in the genome are triggered by pathogen infection. For the analysis of HRF, mutation frequency (MF) and microsatellite instability (MI), we used three different transgenic Arabidopsis lines carrying ß-glucuronidase (GUS)-based substrates in their genome. We found that local infection of Arabidopsis with ORMV resulted in an increase of all three frequencies, albeit to differing degrees. The most prominent increase was observed in microsatellite instability. The increase in HRF was the lowest, although still statistically significant. The analysis of methylation of the 35S promoter and transgene expression showed that the greater instability of the transgene was not attributed to these changes. Strand breaks brought about a significant increase in non-treated tissues of infected plants. The expression of genes associated with various repair processes, such as KU70, RAD51, MSH2, DNA POL α and DNA POL δ, was also increased. To summarize, our data demonstrate that local ORMV infection destabilizes the genome in systemic tissues of Arabidopsis plants in various ways resulting in large rearrangements, point mutations and microsatellite instability.

The importance of the small RNA chaperone Hfq for growth of epidemic Yersinia pestis, but not Yersinia pseudotuberculosis, with implications for plague biology.

Yersinia pestis, the etiologic agent of plague, has only recently evolved from Yersinia pseudotuberculosis. hfq deletion caused severe growth restriction at 37 degrees C in Y. pestis but not in Y. pseudotuberculosis. Strains from all epidemic plague biovars were similarly affected, implicating Hfq, and likely small RNAs (sRNAs), in the unique biology of the plague bacillus.

Hypomethylation and genome instability in the germline of exposed parents and their progeny is associated with altered miRNA expression.

Recent studies suggest that transgenerational genome instability may be epigenetic in nature and mediated via altered DNA methylation and microRNAome. Here, we investigated the nature and mechanisms underlying the disruption of DNA methylation and microRNA expression status in the germline and progeny of exposed parents. We have found that paternal irradiation leads to upregulation of the miR-29 family in the exposed male germline, which causes decreased expression of de novo methyltransferase, DNA methyltransferase 3a, and profound hypomethylation of long interspersed nuclear elements 1 (LINE1) and short interspersed nuclear elements B2 (SINE B2). Epigenetic changes in the male germline further resulted in deleterious effects in the somatic thymus tissue from the progeny of exposed animals, including hypomethylation of LINE1 and SINE B2. Hypomethylation of LINE1 and SINE B2 in the thymus tissue from the progeny was associated with a significant decrease in the levels of lymphoid-specific helicase (LSH) that is crucial for the maintenance of methylation and silencing of repetitive elements. Furthermore, we noted a significant upregulation of miR-468 that targets LSH and leads to its decreased expression in thymus in the progeny of exposed parents. We suggest that miR-468-mediated suppression of LSH leads to aberrant methylation of LINE1 and SINE B2. In summary, altered microRNAome and hypomethylation of retroelements constitute deleterious effects that may significantly influence genome stability of the parental germline and consequently cause genome instability in the progeny.

Alterations of microRNAs and their targets are associated with acquired resistance of MCF-7 breast cancer cells to cisplatin.

Cancer cells that develop resistance to chemotherapeutic agents are a major clinical obstacle in the successful treatment of breast cancer. Acquired cancer chemoresistance is a multifactorial phenomenon, involving various mechanisms and processes. Recent studies suggest that chemoresistance may be linked to drug-induced dysregulation of microRNA function. Furthermore, mounting evidence indicates the existence of similarities between drug-resistant and metastatic cancer cells in terms of resistance to apoptosis and enhanced invasiveness. We studied the role of miRNA alterations in the acquisition of cisplatin-resistant phenotype in MCF-7 human breast adenocarcinoma cells. We identified a total of 103 miRNAs that were overexpressed or underexpressed (46 upregulated and 57 downregulated) in MCF-7 cells resistant to cisplatin. These differentially expressed miRNAs are involved in the control of cell signaling, cell survival, DNA methylation and invasiveness. The most significantly dysregulated miRNAs were miR-146a, miR-10a, miR-221/222, miR-345, miR-200b and miR-200c. Furthermore, we demonstrated that miR-345 and miR-7 target the human multidrug resistance-associated protein 1. These results suggest that dysregulated miRNA expression may underlie the abnormal functioning of critical cellular processes associated with the cisplatin-resistant phenotype.

Chlorine ions but not sodium ions alter genome stability of Arabidopsis thaliana.

Various environmental stresses influence plant genome stability. Most of these stresses, such as ionizing radiation, heavy metals and organic chemicals, represent potent DNA-damaging agents. Here, we show that exposure to NaCl, the stress that is not thought to cause direct DNA damage, results in an increase in the level of strand breaks and homologous recombination rates (RRs) in Arabidopsis thaliana plants. The effect of salt stress on the RR was found to be primarily associated with Cl(-) ions, since exposure of plants to Na(2)SO(4) did not increase the RR, whereas exposure to MgCl(2) resulted in an increase. Changes in the number of strand breaks and in the RR were also paralleled by transcriptional activation of AtRad51 and down-regulation of AtKu70. The progeny of exposed plants exhibited higher RRs, higher expression of AtRad51, lower expression of AtKu70, higher tolerance to salt and methyl methane sulfate (MMS) stresses, as well as a higher increase in RR upon further exposure to stress. Our experiments showed that NaCl is a genotoxic stress that leads to somatic and transgenerational changes in recombination rates, and these changes are primarily triggered by exposure to Cl(-) ions.

Differential sensitivity of Arabidopsis siRNA biogenesis mutants to genotoxic stress.

Plant response to stress has been linked to different RNA-silencing processes and epigenetic mechanisms. Our recent results showed that Arabidopsis thaliana Dicer-like (DCL) mutants were impaired in transgenerational changes, recombination frequency and stress tolerance. We also found that transgenerational changes were dependent on changes in DNA methylation. Here, we hypothesized that plants deficient in the production of small RNAs would show an impaired abiotic stress response. To test this, we exposed A. thaliana dcl2, dcl3, dcl4, dcl2 dcl3 (d2d3), dcl2 dcl4 (d2d4), dcl2 dcl3 dcl4 (d2d3d4), nrpd1a, rdr2 and rdr6 mutants to methyl methane sulfonate (MMS). We found dcl4 and rdr6 to be more sensitive and dcl2, dcl3, d2d3 and rdr2 plants more resistant to MMS, as shown by fresh weight, root length and survival rate. The in vitro repair assay showed the lower ability of dcl2 and dcl3 to repair UV-damaged DNA. To summarize, we found that whereas mutants impaired in transactivating siRNA biogenesis were more sensitive to MMS, mutants impaired in natural antisense siRNA and heterochromatic siRNA biogeneses were more tolerant. Our data suggest that plant response to MMS is in part regulated through biogenesis of various siRNAs.

Differential gene regulation in Yersinia pestis versus Yersinia pseudotuberculosis: effects of hypoxia and potential role of a plasmid regulator.

The molecular basis of the biological differences between Yersinia pestis and Yersinia pseudotuberculosis remains largely unknown, and relatively little is known about environmental regulation of gene expression in these bacteria. We used a proteomic approach to explore the regulatory response of each bacterium to carbon dioxide-supplemented hypoxic conditions. Both organisms responded similarly and the magnitude of their responses was similar to what was observed in low iron conditions. We also identified proteins that were expressed at different levels in Y. pestis and Y. pseudotuberculosis, and found that SodB is expressed more strongly at both the protein and RNA levels in Y. pseudotuberculosis than in Y. pestis. Enzyme activity did not directly correlate with levels of protein expression, and we propose that an amino acid change difference between these orthologous proteins has the potential to affect catalytic activity. In addition, the upstream regulatory regions of several chromosomal genes were found to exhibit specific binding with a putative transcription factor, CDS4, from the Y. pestis-specific pPCPI plasmid. The potential role of this protein in modulating Y. pestis- specific gene regulation warrants further investigation.

Analysis of DNA Hydroxymethylation Using Colorimetric Assay.

Hydroxymethylcytosine (hmC or 5-hmC) is a nitrogen base occurring as a result of cytosine methylation followed by replacing a methyl group with a hydroxyl group through active oxidation. 5-hmC is considered to be one of the forms of epigenetic modification and is suggested as an intermediate step in a semi-active loss of DNA methylation mark. 5-hmC plays an important role in the epigenetic regulation of gene expression in animals, although its role in plants remains controversial. Here, we present a colorimetric method of quantification of 5-hmC using Brassica rapa DNA.

Small RNA Library Preparation and Illumina Sequencing in Plants.

The discovery of small RNAs in plants and animals almost two decades ago attracted a significant interest towards epigenetic regulation of gene expression and the practical implementation of the gained knowledge in applied studies. New and sometimes unexpected functions have been ascribed to sRNAs almost every couple of years since their discovery, hence indicating that the complete role of sRNAs in plant and animal physiology is still barely understood. Next-generation sequencing technologies allow to generate high-resolution profiles of sRNAs for the consequent analysis and possibly to discover novel functions of sRNAs. In this chapter, we provide brief guidelines for sRNA library preparation in plants and a practical approach that can be implemented to overcome possible difficulties with sequencing library generation.

Arabidopsis thaliana siRNA biogenesis mutants have the lower frequency of homologous recombination.

Small interfering RNAs (siRNAs) are involved in the regulation of plant development and response to stress. We have previously shown that mutants impaired in Dicer-like 2 (DCL2), DCL3 and DCL4, RDR2, RDR6 and NPRD1 are partially impaired in their response to stress and dcl2 and dcl3 plants are also impaired in transgenerational response to stress, including changes in homologous recombination frequency (HRF). Here, we have analyzed genome stability of dcl2, dcl3, dcl4, dcl2 dcl3, dcl2 dcl3 dcl4 and rdr6 mutants by measuring the non-induced and the stress-induced recombination frequency. We found that all mutants had the lower spontaneous HRF. The analysis of strand breaks showed that all tested Arabidopsis mutants had a higher level of spontaneous strand breaks, suggesting that the lower HRF is not due to the unusually low level of breaks. Exposure to methyl methane sulfonate (MMS) resulted in an increase in the level of strand breaks in wild-type plants and a decrease in mutants. All mutants had the higher methylation of cytosines at CpG sites under non-induced conditions. Exposure to MMS resulted in a decrease in methylation level in wild-type plants and an increase in methylation in all dcl mutants. The expression of several DNA repair genes was altered in dcl4 plants under non-induced and induced conditions. Our data suggest that siRNA biogenesis may be essential for the maintenance of the genome stability and stress response in Arabidopsis.

Circadian-disruption-induced gene expression changes in rodent mammary tissues.

Evidence is mounting that circadian disruption (CD) is a potential carcinogen in breast cancer development. However, despite the growing concern, to our knowledge, no studies have attempted a genome-wide analysis of CD-induced gene expression changes in mammary tissues. Using a rodent model system, a proven photoperiod-shifting paradigm, varying degrees of CD, and Illumina sequencing, we performed an exploratory genome-wide mRNA analysis in mammary tissues. Even though our analysis did not identify any significant patterns in mRNA levels based on the degree of CD, and the majority of groups did not show changes in gene expression on a large-scale, one group (two-week chronic ZT19) displayed 196 differentially expressed genes, 51 of which have been linked to breast cancer. Through gene-specific pathway analysis, the data illustrate that CD may promote breast cancer development through downregulation of DNA repair and p53 signaling pathways, thus promoting genomic instability and cancer development. Although these results have to be interpreted with caution because only a single group illustrated drastic changes in transcript levels, they indicate that chronic CD may directly induce changes in gene expression on a large-scale with potentially malignant consequences.

Circadian disruption-induced microRNAome deregulation in rat mammary gland tissues.

Breast cancer is the most common malignancy affecting women worldwide, and evidence is mounting that circadian-disruption-induced breast cancer is a warranted concern. Although studies on the role of epigenetics have provided valuable insights, and although epigenetics has been increasingly recognized in the etiology of breast cancer, relatively few studies have investigated the epigenetic link between circadian disruption (CD) and breast cancer. Using a proven photoperiod-shifting paradigm, differing degrees of CD, various tissue-extraction time points, and Illumina sequencing, we investigated the effect of CD on miRNA expression in the mammary tissues of a rodent model system. To our knowledge, our results are the first to illustrate CD-induced changes in miRNA expressions in mammary tissues. Furthermore, it is likely that these miRNA expression changes exhibit varying time frames of plasticity linked to both the degree of CD and length of reentrainment, and that the expression changes are influenced by the light and dark phases of the 24-hour circadian cycle. Of the differentially expressed miRNAs identified in the present study, all but one have been linked to breast cancer, and many have predicted circadian-relevant targets that play a role in breast cancer development. Based on the analysis of protein levels in the same tissues, we also propose that the initiation and development of CD-induced breast cancer may be linked to an interconnected web of increased NF-κB activity and increased levels of Tudor-SN, STAT3, and BCL6, with aberrant CD-induced downregulation of miR-127 and miR-146b potentially contributing to this dynamic. This study provides direct evidence that CD induces changes in miRNA levels in mammary tissues with potentially malignant consequences, thus indicating that the role of miRNAs in CD-induced breast cancer should not be dismissed.

Uncovering genomic differences in human pathogenic Yersinia enterocolitica.

To map out genomic differences between highly pathogenic Yersinia enterocolitica WA-314C biogroup 1B, serotype O:8 strain and low-pathogenic Y. enterocolitica Y-108C biogroup 4, serotype O:3 strain we have applied a method of suppression subtractive hybridization (SSH). In total, 428 WA-314-specific and 83 Y-108-specific sequences were uncovered by SSH. Among them were DNA fragments with similarity to known genes from several groups: (1) genes involved in O-antigen biosynthesis, (2) host-specific restriction-modification systems, (3) systems of iron and heme acquisition and storage, (4) flagellar biogenesis genes, (5) putative virulence factors, (6) drug resistance genes, and (7) mobile elements. Mapped out genomic differences may be applied in identification and development of novel therapeutic strategies for the treatment of enteropathogenic Yersinia.