The hologenome of Daphnia magna reveals possible DNA methylation and microbiome-mediated evolution of the host genome.

Properties that make organisms ideal laboratory models in developmental and medical research are often the ones that also make them less representative of wild relatives. The waterflea Daphnia magna is an exception, by both sharing many properties with established laboratory models and being a keystone species, a sentinel species for assessing water quality, an indicator of environmental change and an established ecotoxicology model. Yet, Daphnia's full potential has not been fully exploited because of the challenges associated with assembling and annotating its gene-rich genome. Here, we present the first hologenome of Daphnia magna, consisting of a chromosomal-level assembly of the D. magna genome and the draft assembly of its metagenome. By sequencing and mapping transcriptomes from exposures to environmental conditions and from developmental morphological landmarks, we expand the previously annotates gene set for this species. We also provide evidence for the potential role of gene-body DNA-methylation as a mutagen mediating genome evolution. For the first time, our study shows that the gut microbes provide resistance to commonly used antibiotics and virulence factors, potentially mediating Daphnia's environmental-driven rapid evolution. Key findings in this study improve our understanding of the contribution of DNA methylation and gut microbiota to genome evolution in response to rapidly changing environments.

A comprehensive epigenomic analysis of phenotypically distinguishable, genetically identical female and male Daphnia pulex.

BACKGROUND: Daphnia species reproduce by cyclic parthenogenesis involving both sexual and asexual reproduction. The sex of the offspring is environmentally determined and mediated via endocrine signalling by the mother. Interestingly, male and female Daphnia can be genetically identical, yet display large differences in behaviour, morphology, lifespan and metabolic activity. Our goal was to integrate multiple omics datasets, including gene expression, splicing, histone modification and DNA methylation data generated from genetically identical female and male Daphnia pulex under controlled laboratory settings with the aim of achieving a better understanding of the underlying epigenetic factors that may contribute to the phenotypic differences observed between the two genders. RESULTS: In this study we demonstrate that gene expression level is positively correlated with increased DNA methylation, and histone H3 trimethylation at lysine 4 (H3K4me3) at predicted promoter regions. Conversely, elevated histone H3 trimethylation at lysine 27 (H3K27me3), distributed across the entire transcript length, is negatively correlated with gene expression level. Interestingly, male Daphnia are dominated with epigenetic modifications that globally promote elevated gene expression, while female Daphnia are dominated with epigenetic modifications that reduce gene expression globally. For examples, CpG methylation (positively correlated with gene expression level) is significantly higher in almost all differentially methylated sites in male compared to female Daphnia. Furthermore, H3K4me3 modifications are higher in male compared to female Daphnia in more than 3/4 of the differentially regulated promoters. On the other hand, H3K27me3 is higher in female compared to male Daphnia in more than 5/6 of differentially modified sites. However, both sexes demonstrate roughly equal number of genes that are up-regulated in one gender compared to the other sex. Since, gene expression analyses typically assume that most genes are expressed at equal level among samples and different conditions, and thus cannot detect global changes affecting most genes. CONCLUSIONS: The epigenetic differences between male and female in Daphnia pulex are vast and dominated by changes that promote elevated gene expression in male Daphnia. Furthermore, the differences observed in both gene expression changes and epigenetic modifications between the genders relate to pathways that are physiologically relevant to the observed phenotypic differences.

Mixtures of Aluminum and Indium Induce More than Additive Phenotypic and Toxicogenomic Responses in Daphnia magna.

Aquatic systems are contaminated by many metals but their effects as mixtures on organisms are not well understood. Here, we assessed effects of aluminum with fairly well-known modes of actions and indium, an understudied emerging contaminant from electronics, followed by studying equi-effective mixtures thereof. We report acute and adverse phenotypic effects in Daphnia magna adults and global transcriptomic effects employing RNA sequencing in neonates. The mixture induced more than additive activity in mortality and in physiological effects, including growth and reproduction. Similarly, transcriptomic effects were more than additive, as indicated by a markedly higher number of 463 differentially expressed transcripts in the mixture and by distinct classes of genes assigned to several biological functions, including metabolic processes, suggesting depleted energy reserves, which may be responsible for the observed impaired reproduction and growth. A gene set enrichment analysis (GSEA) of a priori known response pathways for aluminum confirmed activation of distinct molecular pathways by indium. Our study is highlighting more than additive effects at the transcriptional and physiological level and is providing a state-of-the art approach to mixture analysis, which is important for risk assessment of these metals and metal mixtures.

Use of 5-azacytidine in a proof-of-concept study to evaluate the impact of pre-natal and post-natal exposures, as well as within generation persistent DNA methylation changes in Daphnia.

Short-term exposures at critical stages of development can lead to delayed adverse effects long after the initial stressor has been removed, a concept referred to as developmental origin of adult disease. This indicates that organisms' phenotypes may epigenetically reflect their past exposure history as well as reflecting chemicals currently present in their environment. This concept has significant implications for environmental monitoring. However, there is as yet little or no implementation of epigenetics in environmental risk assessment. In a proof-of-principle study we exposed Daphnia magna to 5-azacytidine, a known DNA de-methylating agent. Exposures covered combinations of prenatal and postnatal exposures as well as different exposure durations and recovery stages. Growth, the transcription of genes and levels of metabolites involved in regulating DNA methylation, and methylation levels of several genes were measured. Our data shows that prenatal exposures caused significant changes in the methylome of target genes, indicating that prenatal stages of Daphnia are also susceptible to same level of change as post-natal stages of Daphnia. While the combination of pre- and postnatal exposures caused the most extreme reduction in DNA methylation compared to the control group. Furthermore, some of the changes in the methylation patterns were persistent even after the initial stressor was removed. Our results suggest that epigenetic biomarkers have the potential to be used as indicators of past chemical exposure history of organisms and provide strong support for implementing changes to the current regimes for chemical risk assessment to mimic realistic environmental scenarios.

Transcriptional and physiological responses induced by binary mixtures of drospirenone and progesterone in zebrafish (Danio rerio).

Drospirenone (DRS) is a synthetic progestin increasingly used in oral contraceptives with similar effects to progesterone (P4). Wild fish are exposed to DRS and P4 through wastewater. However, the effects of DRS on fish, both as an individual compound and in mixtures, have not been extensively studied. Therefore, in this study, global gene expression profiles of ovary and brain of female zebrafish (Danio rerio) were characterized after exposure to 55, 553, and 5442 ng/L DRS for 14 days. The effects were then compared to the observed responses after exposure to mixtures of DRS and P4 (DRS+P4: 27 + 0.8, 277 + 8 and 3118 + 123 ng/L). Transcriptomics findings were related to the changes in vitellogenin protein concentrations in the blood, morphology, and histology of gonads. Multivariate analysis indicated tissue-, dose-, and treatment-dependent expression profiles. Genes involved in steroid hormone receptor activity and circadian rhythm were enriched in DRS and mixture groups, among other pathways. In mixtures, the magnitude of response was dose- and transcript-dependent, both at the molecular and physiological levels. Effects of DRS and P4 were additive for most of the investigated parameters and occurred at environmentally relevant concentrations. They may translate to adverse reproductive effects in fish.

Designing an Interdisciplinary Health Course: A Qualitative Study of Undergraduate Students' Experience of Interdisciplinary Curriculum Design and Learning Experiences.

OBJECTIVES: Research into interdisciplinary education, where concrete examples and empirical evidence of interdisciplinary teaching is explored, is limited. Furthermore, there are no standardized guidelines on best practices for designing and implementing an interdisciplinary curriculum. Recently, in healthcare settings there has been a drive to adopt interdisciplinary or transdisciplinary practices, creating a need for training individuals capable of working across discipline-specific boundaries, or to even adopt a transdisciplinary practice. This is partially attributed to recognizing that local and global complex health challenges are interlinked and share common factors and often require a new integrated approach to management. In response, a new interdisciplinary course using a modified snowflake model of interdisciplinary course design was launched at a medical school. The course aimed to provide a broad foundation for lifelong learning with a strong emphasis on the development of knowledge, skills, and professional values essential for interdisciplinary and transdisciplinary practice in applied health promotion for individuals and society. METHODS: A semi-structured focus group with students (n = 15% of the inaugural cohort) having completed at least 1 year of the course was undertaken to investigate student perspectives on best approaches for the development and delivery of interdisciplinary learning and teaching. RESULTS: Results highlighted the importance of providing training and opportunity for students to practice integration within the curriculum. Additionally, it was noted that including a module to introduce students to different disciplines and guiding students to explore their inherent interconnectedness is essential in helping them develop interdisciplinary thinking and skills. Crucially, the role of integrated assessments was also recognized as fundamental for demonstrating and practicing interdisciplinarity. CONCLUSION: Overall, this study provides valuable insights and recommendations for educators with the objective of developing interdisciplinary learning in new or existing higher education courses or those seeking to prepare learners for contemporary and emergent societal challenges more generally.

Disruption of DNA methylation via S-adenosylhomocysteine is a key process in high incidence liver carcinogenesis in fish.

Interactions between epigenome and the environment in biology and in disease are of fundamental importance. The incidence of hepatocellular adenomas in flatfish exceeds 20% in some environments forming a unique opportunity to study environmental tumorigenesis of general relevance to cancer in humans. We report the novel finding of marked DNA methylation and metabolite concentration changes in histopathologically normal tissue distal to tumors in fish liver. A multi-"omics" discovery approach led to targeted and quantitative gene transcription analyses and metabolite analyses of hepatocellular adenomas and histologically normal liver tissue in the same fish. We discovered a remarkable and consistent global DNA hypomethylation, modification of DNA methylation and gene transcription, and disruption of one-carbon metabolism in distal tissue compared to livers of non-tumor-bearing fish. The mechanism of this disruption is linked not to depletion of S-adenosylmethionine, as is often a feature of mammalian tumors, but to a decrease in choline and elevated S-adenosylhomocysteine, a potent inhibitor of DNA methyltransferase. This novel feature of normal-appearing tissue of tumor-bearing fish helps to understand the unprecedentedly high incidence of tumors in fish sampled from the field and adds weight to the controversial epigenetic progenitor model of tumorigenesis. With further studies, the modifications may offer opportunities as biomarkers of exposure to environmental factors influencing disease.

Comprehensive profiling of zebrafish hepatic proximal promoter CpG island methylation and its modification during chemical carcinogenesis.

BACKGROUND: DNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis. RESULTS: A novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver. DNA methylation alterations were detected in zebrafish liver tumors induced by the environmental carcinogen 7, 12-dimethylbenz(a)anthracene. Genes significantly hypomethylated in tumors were associated particularly with proliferation, glycolysis, transcription, cell cycle, apoptosis, growth and metastasis. Hypermethylated genes included those associated with anti-angiogenesis and cellular adhesion. Of 49 genes that were altered in expression within tumors, and which also had appropriate CpG islands and were co-represented on the tiling array, approximately 45% showed significant changes in both gene expression and methylation. CONCLUSION: The functional pathways containing differentially methylated genes in zebrafish hepatocellular carcinoma have also been reported to be aberrantly methylated during tumorigenesis in humans. These findings increase the confidence in the use of zebrafish as a model for human cancer in addition to providing the first comprehensive mapping of DNA methylation in the normal adult zebrafish liver.

Characterisation of the dynamic nature of lipids throughout the lifespan of genetically identical female and male Daphnia magna.

Lipids play a significant role in regulation of health and disease. To enhance our understanding of the role of lipids in regulation of lifespan and healthspan additional studies are required. Here, UHPLC-MS/MS lipidomics was used to measure dynamic changes in lipid composition as a function of age and gender in genetically identical male and female Daphnia magna with different average lifespans. We demonstrate statistically significant age-related changes in triglycerides (TG), diglycerides (DG), phosphatidylcholine, phosphatidylethanolamine, ceramide and sphingomyelin lipid groups, for example, in males, 17.04% of TG lipid species decline with age whilst 37.86% increase in relative intensity with age. In females, 23.16% decrease and 25.31% increase in relative intensity with age. Most interestingly, the rate and direction of change can differ between genetically identical female and male Daphnia magna, which could be the cause and/or the consequence of the different average lifespans between the two genetically identical genders. This study provides a benchmark dataset to understand how lipids alter as a function of age in genetically identical female and male species with different average lifespan and ageing rate.

Ageing differently: Sex-dependent ageing rates in Daphnia magna.

Ageing is defined as the gradual decline of normal physiological functions in a time-dependent manner. Significant progress has been made in characterising the regulatory processes involved in the mechanisms of ageing which would have been hindered without the use of model organisms. Use of alternative model organisms greatly diversifies our understanding of different factors underpinning the ageing process and the potential translation for human application. Unique characteristics make Daphnia an attractive model organism for research into mechanisms underlying ageing, such as transparent body, short generation time, well-characterised methylome, regenerative capabilities and available naturally occurring ecotypes. Most interestingly, genetically identical female and male Daphnia have evolved different average lifespans, providing a unique opportunity for understanding the underlying mechanisms of ageing and regulation of lifespan. Investigating sex differences in longevity could provide insight into principal mechanisms of ageing and lifespan regulation. In this study we provide evidence in support of establishing genetically identical female and male Daphnia as unique and valuable resources for research into mechanisms of ageing and begin to delineate the mechanisms involved in sex differences in lifespan. We identify significant differences between genders in physiological markers such as lifespan, growth rate, heart rate and swimming speed in addition to molecular markers such as lipid peroxidation product accumulation, thiol content decline and age-dependent decline in DNA damage repair efficiency. Overall, our data indicates that investigating sex differences in longevity in the clonal organism Daphnia under controlled laboratory conditions can provide insight into principal mechanisms of ageing and lifespan regulation.

Major trauma and acceleration of the ageing process.

It is well established that numerous factors can affect the rate at which we age biologically. Diet, physical activity, lifestyle and our genes all play a major role in influencing the ageing trajectory and longevity. Major trauma affects millions globally, is the major cause of death in young adults and could influence ageing processes but has largely been ignored by biogenterologists. The long-term health consequences of physical trauma are well known in the medical community, how trauma effects the ageing process at a molecular level is not. It has long been difficult to assess ageing trajectories due to the absence of a biomarker of biological rather than chronological age. Recent advances in epigenetics have helped by identifying specific DNA methylation sites as good indicators of biological age. Recent investigations into the impact of psychological trauma and the associated physical stress on accelerating ageing as measured by epigenetic drift are promising. The physical and metabolic stress which is synonymous with physical trauma may also accelerate the ageing process. We suggest that long term epigenetic profiling is required to understand to what degree the ageing trajectory is altered by trauma, which will in turn add support for the development of novel therapies to improve health outcomes for survivors of traumatic injury.

Pattern of DNA Methylation in Daphnia: Evolutionary Perspective.

DNA methylation is an evolutionary ancient epigenetic modification that is phylogenetically widespread. Comparative studies of the methylome across a diverse range of non-conventional and conventional model organisms is expected to help reveal how the landscape of DNA methylation and its functions have evolved. Here, we explore the DNA methylation profile of two species of the crustacean Daphnia using whole genome bisulfite sequencing. We then compare our data with the methylomes of two insects and two mammals to achieve a better understanding of the function of DNA methylation in Daphnia. Using RNA-sequencing data for all six species, we investigate the correlation between DNA methylation and gene expression. DNA methylation in Daphnia is mainly enriched within the coding regions of genes, with the highest methylation levels observed at exons 2-4. In contrast, vertebrate genomes are globally methylated, and increase towards the highest methylation levels observed at exon 2, and maintained across the rest of the gene body. Although DNA methylation patterns differ among all species, their methylation profiles share a bimodal distribution across the genomes. Genes with low levels of CpG methylation and gene expression are mainly enriched for species specific genes. In contrast, genes associated with high methylated CpG sites are highly transcribed and evolutionary conserved across all species. Finally, the positive correlation between internal exons and gene expression potentially points to an evolutionary conserved mechanism, whereas the negative regulation of gene expression via methylation of promoters and exon 1 is potentially a secondary mechanism that has been evolved in vertebrates.

Optimisation of DNA extraction from the crustacean Daphnia.

Daphnia are key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA from Daphnia spp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that if Daphnia magna are homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin in Daphnia's carapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses.

Epigenetic memory of environmental organisms: a reflection of lifetime stressor exposures.

Both genetic and epigenetic responses of organisms to environmental factors, including chemical exposures, influence adaptation, susceptibility to toxicity and biodiversity. In model organisms, it is established that epigenetic alterations, including changes to the methylome, can create a memory of the received signal. This is partly evidenced through the analysis of epigenetic differences that develop between identical twins throughout their lifetime. The epigenetic marks induce alterations to the gene expression profile, which, in addition to mediating homeostatic responses, have the potential to promote an abnormal physiology either immediately or at a later stage of development, for example leading to an adult onset of disease. Although this has been well established, epigenetic mechanisms are not considered in chemical risk assessment or utilised in the monitoring of the exposure and effects of chemicals and environmental change. In this review, epigenetic factors, specifically DNA methylation, are highlighted as mechanisms of adaptation and response to environmental factors and which, if persistent, have the potential, retrospectively, to reflect previous stress exposures. Thus, it is proposed that epigenetic "foot-printing" of organisms could identify classes of chemical contaminants to which they have been exposed throughout their lifetime. In some cases, the potential for persistent transgenerational modification of the epigenome may also inform on parental germ cell exposures. It is recommended that epigenetic mechanisms, alongside genetic mechanisms, should eventually be considered in environmental toxicity safety assessments and in biomonitoring studies. This will assist in determining the mode of action of toxicants, no observed adverse effect level and identification of biomarkers of toxicity for early detection and risk assessment in toxicology but there are critical areas that remain to be explored before this can be achieved.

The toxicological application of transcriptomics and epigenomics in zebrafish and other teleosts.

Zebrafish (Danio rerio) is one of a number of teleost fish species frequently employed in toxicology. Toxico-genomics determines global transcriptomic responses to chemical exposures and can predict their effects. It has been applied successfully within aquatic toxicology to assist in chemical testing, determination of mechanisms and environmental monitoring. Moreover, the related field of toxico-epigenomics, that determines chemical-induced changes in DNA methylation, histone modifications and micro-RNA expression, is emerging as a valuable contribution to understanding mechanisms of both adaptive and adverse responses. Zebrafish has proven a useful and convenient model species for both transcriptomic and epigenetic toxicological studies. Despite zebrafish's dominance in other areas of fish biology, alternative fish species are used extensively in toxico-genomics. The main reason for this is that environmental monitoring generally focuses on species native to the region of interest. We are starting to see advances in the integration of high-throughput screening, omics techniques and bioinformatics together with more traditional indicator endpoints that are relevant to regulators. Integration of such approaches with high-throughput testing of zebrafish embryos, leading to the discovery of adverse outcome pathways, promises to make a major contribution to ensuring the safety of chemicals in the environment.

Impacts of TCDD and MeHg on DNA methylation in zebrafish (Danio rerio) across two generations.

This study aimed to investigate whether dioxin (TCDD) and methylmercury (MeHg) pose a threat to offspring of fish exposed to elevated concentrations of these chemicals via epigenetic-based mechanisms. Adult female zebrafish were fed diets added either 20 µg/kg 2,3,7,8 TCDD or 10 mg/kg MeHg for 47 days, or 10 mg/kg 5-aza-2'-deoxycytidine (5-AZA), a hypomethylating agent, for 32 days, and bred with unexposed males in clean water to produce F1 and F2 offspring. Global DNA methylation, promoter CpG island methylation and target gene transcription in liver of adult females and in 3 days post fertilization (dpf) F1 and F2 embryos were determined with HPLC, a novel CpG island tiling array containing 54,933 different probes and RT-qPCR, respectively. The results showed that chemical treatment had no significant effect on global DNA methylation levels in F1 (MeHg and TCDD) and F2 (MeHg) embryos and only a limited number of genes were identified with altered methylation levels at their promoter regions. CYP1A1 transcription, an established marker of TCDD exposure, was elevated 27-fold in F1 embryos compared to the controls, matching the high levels of CYP1A1 expression observed in F0 TCDD-treated females. This suggests that maternal transfer of TCDD is a significant route of exposure for the F1 offspring. In conclusion, the selected doses of TCDD and MeHg, two chemicals often found in high concentrations in fish, appear to have only modest effects on DNA methylation in F1 (MeHg and TCDD) and F2 (MeHg) embryos of treated F0 females.

Gene expression and epigenetic changes by furan in rat liver.

Furan, a widely used industrial compound, has been found in a number of heated food items. Furan is carcinogenic to rats and mice, but the mechanism behind its carcinogenic effect is still not well understood. In this study, we tested the hypothesis that alteration of gene expression relating to cell cycle, apoptosis, DNA damage and of epigenetic modifications including miRNA and DNA methylation may contribute to rodent carcinogenicity of furan. Using quantitative PCR arrays specific to cell cycle-, apoptosis- and DNA damage-related genes, we found that three months furan treatment at 30 mg/kg (5 daily doses per week) induced extensive mRNA expression changes (largely up-regulation) in male Sprague Dawley rat liver, and the gene expression changes did not fully recover after a one month withdrawal of furan. We also found 18 miRNAs were up-regulated and 12 were down-regulated by PCR arrays. Many of these deregulated miRNAs were also found to have similar changes in furan-induced tumour samples. Both hyper- and hypo-methylation of specific gene promoter regions were identified and validated in the 3-month samples and tumour samples by microarray and COBRA (combined bisulfite restriction analysis). No global DNA methylation change was found in the 3 month treatment groups by LC-MS/MS, while furan-induced tumour samples showed global hypomethylation compared to non-tumour tissues. In conclusion, three months furan treatment at a carcinogenic dose resulted in irreversible gene expression changes, miRNA modulation and DNA methylation alteration in combination with a DNA-damage response, which suggests that non-genotoxic mechanisms are important for furan carcinogenicity.

DNA methylation in liver tumorigenesis in fish from the environment.

The link between environment, alteration in DNA methylation and cancer has been well established in humans; yet, it is under-studied in unsequenced non-model organisms. The occurrence of liver tumors in the flatfish dab collected at certain UK sampling sites exceeds 20%, yet the causative agents and the molecular mechanisms of tumor formation are not known, especially regarding the balance between epigenetic and genetic factors. Methylated DNA Immunoprecipitation (MeDIP) combined with de novo high-throughput DNA sequencing were used to investigate DNA methylation changes in dab hepatocellular adenoma tumors for the first time in an unsequenced species. Novel custom-made dab gene expression arrays were designed and used to determine the relationship between DNA methylation and gene expression. In addition, the confirmatory techniques of bisulfite sequencing PCR (BSP) and RT-PCR were applied. Genes involved in pathways related to cancer, including apoptosis, wnt/ß-catenin signaling and genomic and non-genomic estrogen responses, were altered both in methylation and transcription. Global methylation was statistically significantly 1.8-fold reduced in hepatocellular adenoma and non-cancerous surrounding tissues compared with liver from non-cancer bearing dab. Based on the identified changes and chemical exposure data, our study supports the epigenetic model of cancer. We hypothesize that chronic exposure to a mixture of environmental contaminants contributes to a global hypomethylation followed by further epigenetic and genomic changes. The findings suggest a link between environment, epigenetics and cancer in fish tumors in the wild and show the utility of this methodology for studies in non-model organisms.

Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells.

An abundant form of DNA damage caused by reactive oxygen species is 8-oxo-7,8-dihydroguanine for which the base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) is a major repair enzyme. To assess the location and intracellular activity of the OGG1 protein in response to oxidative stress, we have utilised a fluorescence-quench molecular beacon switch containing a 8-oxo-dG:C base pair and a fluorescent and quencher molecule at opposite ends of a hairpin oligonucleotide. Oxidative stress was induced by treatment with potassium bromate. Flow cytometry demonstrated a concentration-dependent increase in the activity of OGG1 that was detected by the fluorescence produced when the oligonucleotide was cleaved in the cells treated with potassium bromate. This signal is highly specific and not detectable in OGG1 knock out cells. Induction of OGG1 activity is not a result of induction of OGG1 gene expression as assessed by qPCR suggesting a role for protein stabilisation or increased OGG1 catalytic activity. High resolution confocal microscopy pinpointed the location of the fluorescent molecular beacon in live cells to perinuclear regions that were identified as mitochondria by co-staining with mitotracker dye. There is no evidence of cut beacon within the nuclear compartment of the cell. Control experiments with a positive control beacon (G:C base pair and lacking the DAB quencher) did not result in mitochondrial localisation of fluorescence signal indicating that the dye does not accumulate in mitochondria independent of OGG1 activity. Furthermore, faint nuclear staining was apparent confirming that the beacon structure is able to enter the nucleus. In conclusion, these data indicate that the mitochondria are the major site for OGG1 repair activity under conditions of oxidative stress.