Changes In DNA Methylation Levels Research Articles

Changes in DNA methylation levels during seed development in Jatropha curcas.

Changes in DNA methylation levels during seed development in Jatropha curcas.

DNA Methylation and Chromatin Regulation during Fleshy Fruit Development and Ripening.

Fruit ripening is a developmental process that results in the leaf-like carpel organ of the flower becoming a mature ovary primed for dispersal of the seeds. Ripening in fleshy fruits involves a profound metabolic phase change that is under strict hormonal and genetic control. This work reviews recent developments in our understanding of the epigenetic regulation of fruit ripening. We start by describing the current state of the art about processes involved in histone post-translational modifications and the remodeling of chromatin structure and their impact on fruit development and ripening. However, the focus of the review is the consequences of changes in DNA methylation levels on the expression of ripening-related genes. This includes those changes that result in heritable phenotypic variation in the absence of DNA sequence alterations, and the mechanisms for their initiation and maintenance. The majority of the studies described in the literature involve work on tomato, but evidence is emerging that ripening in other fruit species may also be under epigenetic control. We discuss how epigenetic differences may provide new targets for breeding and crop improvement.

Epigenetics and cortical spreading depression: changes of DNA methylation level at retrotransposon sequences

Cortical spreading depression (CSD) is an evolutionarily conserved phenomenon that involves a slow and self-propagating depolarization wave associated with spontaneous depression of electrical neuronal activity. CSD plays a central role in the pathophysiology of several brain diseases and is considered to be able to promote "Preconditioning". This phenomenon consists of the brain protecting itself against future injury by adaptation. Understanding of the molecular mechanisms underlying Preconditioning has significant clinical implications. We have already proposed that the long-lasting effects of CSD could be related to silencing of retrotransposon sequences by histone methylation. We analyzed DNA methylation of two retrotransposon sequences, LINE1 and L1, and their corresponding expression pattern after CSD induction. Based on immunoprecipitation assay of the methylated DNA (meDIP), we demonstrated hypermethylation of both sequences in preconditioned rat brain cortex compared with a control 24h after CSD induction. Using quantitative PCR, we also showed that CSD induction caused a decrease of the transcript level of both retrotransposon sequences. Our data are consistent with the hypothesis of epigenetic modifications in Preconditioning-dependent neuroprotection by increasing genome stability via the silencing of retrotransposon sequences.

Metabolic and Transcriptional Changes in Cultured Muscle Stem Cells from Low Birth Weight Subjects.

Developmental programming of human muscle stem cells could in part explain why individuals born with low birth weight (LBW) have an increased risk of developing type 2 diabetes (T2D) later in life. We hypothesized that immature muscle stem cell functions including abnormal differentiation potential and metabolic function could link LBW with the risk of developing T2D. Design/Settings/Participants: We recruited 23 young men with LBW and 16 age-matched control subjects with normal birth weight. Biopsies were obtained from vastus lateralis, and muscle stem cells were isolated and cultured into fully differentiated myotubes. We studied glucose uptake, glucose transporters, insulin signaling, key transcriptional markers of myotube maturity, selected site-specific DNA methylation, and mitochondrial gene expression. We found reduced glucose uptake as well as decreased levels of glucose transporter-1 and -4 mRNA and of the Akt substrate of 160-kDa mRNA and protein in myotubes from LBW individuals compared with normal birth weight individuals. The myogenic differentiation markers, myogenin and myosin heavy chain 1 and 2, were decreased during late differentiation in LBW myotubes. Additionally, mRNA levels of the peroxisome proliferator-activated receptor-γ coactivator-1α and cytochrome c oxidase polypeptide 7A were reduced in LBW myotubes. Decreased gene expression was not explained by changes in DNA methylation levels. We demonstrate transcriptional and metabolic alterations in cultured primary satellite cells isolated from LBW individuals after several cell divisions, pointing toward a retained intrinsic defect conserved in these myotubes.

Genetic Studies on Mammalian DNA Methyltransferases.

Cytosine methylation at the C5-position, generating 5-methylcytosine (5mC), is a DNA modification found in many eukaryotic organisms, including fungi, plants, invertebrates, and vertebrates, albeit its levels vary greatly in different organisms. In mammals, cytosine methylation occurs predominantly in the context of CpG dinucleotides, with the majority (60-80 %) of CpG sites in their genomes being methylated. DNA methylation plays crucial roles in the regulation of chromatin structure and gene expression and is essential for mammalian development. Aberrant changes in DNA methylation levels and patterns are associated with various human diseases, including cancer and developmental disorders. DNA methylation is mediated by three active DNA methyltransferases (Dnmts), namely, Dnmt1, Dnmt3a, and Dnmt3b, in mammals. Over the last two decades, genetic manipulations of these enzymes, as well as their regulators, in mice have greatly contributed to our understanding of the biological functions of DNA methylation in mammals. In this chapter, we discuss genetic studies on mammalian Dnmts, focusing on their roles in embryogenesis, cellular differentiation, genomic imprinting, and X-chromosome inactivation.

Changes in methylation of genomic DNA from chicken immune organs in response to H5N1 influenza virus infection.

DNA methylation is an important epigenetic modification in eukaryotes, which plays a significant role in regulating gene expression. When the host is invaded by the influenza virus, gene expression is regulated via changes in DNA methylation levels or patterns, leading to the activation or suppression of relevant signaling pathways or networks, triggering a series of immune responses against viral invasion. Here, we investigated the changes in genomic DNA methylation in the immune organs of chicken infected with H5N1 influenza virus. Genome-wide DNA methylation levels in the spleen, thymus, and bursa of Fabricius of specific pathogen-free (SPF) chicken infected with the Guangdong (G-H5N1) and Anhui (A-H5N1) H5N1 strains, and water (control) were analyzed by fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP). The results indicated that total DNA methylation levels did not differ between spleen genomic DNA in chicken treated with different viral strains and the control (P > 0.05). However, the total DNA methylation levels were significantly upregulated in the thymus (P < 0.01) and bursa (P < 0.05) of chicken in the A-H5N1 group compared to those in the G-H5N1 and control groups. These results provide a basis for the screening of avian influenza-resistance genes or methylation markers, analyzing the epigenetic regulation mechanisms of avian influenza, and performing selective breeding for disease resistance.

P88: Multiple mechanisms of DNA minor groove binding ligand epigenetic effects

Purpose DNA minor groove is the main target of small molecules, which noncovalently and to a certain extent site-specifically bind to appropriate nucleotide sequences. Study of these substances can give rise to understanding the mechanistic relationship between sites of interaction and activity of appropriate enzymes with “houskeeping” function including helicases, topoisomerases, methyltransferases, demethylases and DNA/RNA-polymerases. Results We revealed for the first time that AT-specific minor groove binding ligands (MGBLs), in particular bisbenzimidazoles (Hoechst33258 and its derivatives), widely used in molecular and cell biology for DNA-staining, induce loss of heterozygosity at high frequency while point mutations and chromosome deletions at insignificant levels. Moreover, we demonstrated that the agents realized their genotoxic blastomogenic effects via homologous recombination mechanism exclusively. Lately the same mechanism of genotoxicity has been shown for MGBL carbazole derivative Curaxin, which is toxic for a broad range of tumor cell lines in vitro and inhibit tumor growth in different mouse models of cancer in vivo. Moreover, powerful antitumor activity has been demonstrated for Trabectedin, which binds to the DNA’s minor groove and alkylate guanine residues. All this provided a framework for wide-ranging investigation of cell response to MGBLs exposure, molecular mechanisms of their recombinogenic as also their anticancer activity. A special interest is paid to epigenetic mechanisms of MGBs action. Our study aimed to examine the epigenetic effects of recombinogenic (Hoechst33342, Hoechst33258) and non-recombinogenic (DAPI, Diminazene, Pentamidine and Netropsin) MGBLs. After we unmasked MGBLs’ recombinogenic activity, we hypothesized that their molecular mechanism of indirect DNA damage involves poly(ADP-ribose)polymerase-1 (PARP-1) activation. Surprisingly, we found that all AT-specific MGBLs preventing PARP-1 interaction with DNA inhibit its activation, and hence, the DNA-dependent pathway of PARP-1 activation function. These inhibitors effectively block PARP-1 activity in vivo, as it was demonstrated in a Drosophila experimental system and in human breast cancer-derived BT474 cell line. Further epigenetic effects of these indirect genotoxic carcinogens were analyzed using HeLa cell population with epigenetically suppressed GFP-reporter gene as a model. All compounds had strong GFP- reactivation effect. The obtained results confirm scarce data of previous publications on the ability of DNA minor groove ligands to influence gene transcription process. Statistically significant results of changes of DNA methylation level were detected under 5-azaC and Hoechst 33258 treatment, but it was absent after Hoechst 33342 treatment. For the rest compounds significant loss of promoter region methylation was not observed. The common epigenetic marks of transcription include histone H3 trimethylation in lysine 4 (H3K4me3) and histone H3 and H4 acetylation (acH3/acH4) on promoter regions of genes. We showed that TSA, Hoechst 33342 and DAPI treatment of HeLa-TI cells lead to increased level of histone H3 trimethylation of lysines 4 (H3K4me3), but for all that the level of histone H4 acetylation remains without significant changes. On the contrary, histone H3 acetylation level was stably increased at all samples. The modifications are typical for silent genes and decrease of their amount suggests the transcriptional reactivation. Loss of H4K20me3 mark in comparison with untreated control was demonstrated for MGBLs. Conclusion Taking together, findings of the study will lay the fundamental groundwork for the development of novel anticancer strategies and new chemotherapy effects of small molecules. The mechanism of PARP-1 inhibition by MGBLs and its epigenetic influence on silent genes, via DNA methylation and histone modifications make reasonable further study of these compounds in three prospective directions: (1) as self-acting cytotoxic agents; (2) as a component of combined chemotherapy targeting DNA repair by PARP-1, thereby facilitating DNA damage caused by other anticancer drugs; (3) as an agents reactivating epigenetically repressed genes which silencing occurs during the earliest stages of neoplasia and accumulates with progression toward malignancy.

Epigenetic instability of imprinted genes in human cancers.

Many imprinted genes are often epigenetically affected in human cancers due to their functional linkage to insulin and insulin-like growth factor signaling pathways. Thus, the current study systematically characterized the epigenetic instability of imprinted genes in multiple human cancers. First, the survey results from TCGA (The Cancer Genome Atlas) revealed that the expression levels of the majority of imprinted genes are downregulated in primary tumors compared to normal cells. These changes are also accompanied by DNA methylation level changes in several imprinted domains, such as the PEG3, MEST and GNAS domains. Second, these DNA methylation level changes were further confirmed manually using several sets of cancer DNA. According to the results, the Imprinting Control Regions of the PEG3, MEST and GNAS domains are indeed affected in breast, lung and ovarian cancers. This DNA methylation survey also revealed that evolutionarily conserved cis-regulatory elements within these imprinted domains are very variable in both normal and cancer cells. Overall, this study highlights the epigenetic instability of imprinted domains in human cancers and further suggests its potential use as cancer biomarkers.

Effects of DNA methylation inhibitors and conventional antidepressants on mice behaviour and brain DNA methylation levels.

Stress increases DNA methylation and decreases the expression of genes involved in neural plasticity, while treatment with DNA methyltransferase inhibitors (DNMTi) increases gene expression and induces antidepressant-like effects in preclinical models. Therefore, the aim of the present work was to further investigate the potential antidepressant-like effect induced by DNMTi by evaluating the behavioural effects induced by associating DNMTi treatment with conventional antidepressant drugs in mice submitted to the forced swimming test (FST). In addition, brain levels of DNA methylation were also investigated. Mice received systemic injections of 5-aza-2'-deoxycytidine (5-AzaD, 0.1, 0.2 mg/kg), RG108 (0.1, 0.2, 0.4 mg/kg), desipramine (DES, 2.5, 5, 10 mg/kg) or fluoxetine (FLX, 5, 10, 20, 30 mg/kg) and were submitted to the FST or to the open field test (OFT). Additional groups received a combination of subeffective doses of 5-AzaD or RG108 (DNMTi) with subeffective doses of DES or FLX (antidepressants). Subeffective doses of RG108 (0.1 mg/kg) or 5-AzaD (0.1 mg/kg) in association with subeffective doses of DES (2.5 mg/kg) or FLX (10 mg/kg) induced significant antidepressant-like effects. Effective doses of RG108 (0.2 mg/kg), 5-AzaD (0.2 mg/kg), DES (10 mg/kg) and FLX (20 mg/kg) atenuated stress-induced changes in DNA methylation levels in the hippocampus and prefrontal cortex. None of the treatments induced locomotor effects in the OFT. These results suggest that DNMTi potentiate the behavioural effects of antidepressant drugs in the FST and that antidepressants, as well as DNMTi, are able to modulate stress-induced changes in DNA methylation in brain regions closely associated with the neurobiology of depression.

DNA hypomethylation concomitant with the overproduction of ROS induced by naphthoquinone juglone on tobacco BY-2 suspension cells

Juglone, 5-hydroxy-1,4-naphthoquinone, is a naturally occurring plant secondary metabolite with allelopathic and cytotoxic properties. The cytotoxic effect of juglone and changes at the level of DNA methylation were investigated on a plant cell model, tobacco BY-2 cell suspension culture. We found that juglone in a concentration-dependent manner inhibits cell growth and causes creation of reactive oxygen species (ROS) that were visible mainly in nucleus, nucleolus, cytoplasm, and plasma membrane. Analysis of the changes in DNA methylation level using Luminometric Methylation Assay showed that juglone at the concentrations of 60 and 80μM causes hypomethylation of DNA. Decrease in DNA methylation was confirmed by the more sensitive technique, confocal immunofluorescence microscopy, already at 30μM, and was accompanied by significant overproduction of hydrogen peroxide, hydroxyl radicals, and superoxide anion radicals. The highest percentage of the signs of programed cell death, especially apoptic-like bodies that indicate DNA fragmentation was observed at 30μM of juglone. On the basis of results we suggest that overproduction of ROS is the main mechanism that causes hypomethylation of DNA, which may contribute to the initiation of the programmed cell death.

Changes of DNA Methylation Levels and Patterns in Tea Plant ( Camellia sinensis ) during Cold Acclimation

Low temperature is one of the most critical environmental factors that limit tea plant growth, survival and geographical distribution. Tea plant can enhance its cold tolerance after undergoing a period of cold acclimation. DNA methylation is one of the epigenetic phenomena in plant, and can be altered by environmental stress. In order to explore the relationship between DNA me- thylation and low temperature stress response in tea plant, methylation sensitive amplified polymorphism (MSAP) and high per- formance liquid chromatography (HPLC) were used to analyze the changes of DNA methylation level and pattern in this study. The MSAP results showed that 905, 968, and 970 methylated bands were amplified with 50 selected primers in non-acclimated sample (CK), fully acclimated and de-acclimated samples, with the methylation levels of 50.6%, 54.1%, and 54.2%, respectively. DNA methylation levels in fully acclimated and de-acclimated samples were increased compared with CK. HPLC results were similar with MSAP results. In addition, DNA demethylation and methylation were both occurred during cold acclimation, but the DNA methylation level was increased more than the DNA demethylation level. These results indicated that DNA methylation may participate in the response of cold acclimation in tea plant.

Analysis of CpG Island DNA Methylation of p15INK4b and RIL in Bone Marrow Samples of Patients with Monoclonal Gammopathies

Introduction: Adding a methyl group on the cytosine residues of CpG dinucleotides (CpGs) is a basic phenomenon in DNA methylation. This modification belongs to a group of epigenetic changes that are thought to play one of the key roles in tumor development and progression. DNA hypermethylation occurs mainly in CpG islands of the promoter region in tumor-supressor (TS) genes, which leads to gene silencing. On the other hand, global DNA hypomethylation leads to a genomic instability. Many specific genes are epigenetically changed in individual malignant diseases and these genes are intensively studied by the scientific community. According to the recent research in plasma cell neoplasms, the global genic hypomethylation is predominant in contrast to hypermethylation events. Epigenetic changes of TS genes may worsen prognosis, drug response and microenvironment interaction in multiple myeloma patients. Our aim was to assess the methylation level of the RIL and p15INK4b gene. RIL protein acts as suppressor of cell proliferation and it sensitizes tumor cells to apoptosis, on the other hand, the p15INK4bplays an important role in inhibiting the cell cycle progression in G1 phase.Methods: Our updated study includes 72 bone marrow aspirate-samples from 68 patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS). 68 samples were acquired at the time of the diagnosis and four patients with MM were assessed also in remission after chemotherapy. Extracted DNA after bisulfite modification was examined by pyrosequencing method (Pyromark Q96, Qiagen, Germany) and the selected gene regions – RIL promotor, p15INK4b promotor and p15INK4b exon were analysed. Nine CpGs in the RIL promoter, thirteen CpGs in the p15INK4b promoter and sixteen CpGs in the p15INK4bexon were studied. Average methylation level (MtL) was generated and samples were sorted according to the mean MtL (%) into four groups for the RIL gene (less than 10% MtL, 11-19% MtL, 21-50% and more than 50%). We used commercially available unmethylated DNA and methylated DNA control. Statistical analysis was performed using Pyromark Software and Wilcoxon rank sum test.Results: Out of the four groups for the methylation level of the RIL gene promoter, the groups with 11-19% MtL and 21-50% MtL, detected significant and highly significant differences respectively at both p-values - p-value ≤0.05 and p-value ≤0.01. There were only 2 samples with MtL level over 50% in patients with active MM, precluding valid statistical analysis. In both regions of the p15INK4b gene, i.e. the promoter and the exon, we did not find any significant differences between the methylation levels - mean MtL ranged from 3% to 6% in p15INK4b promotor and from 3% to 12% in p15INK4b exon. The time-dependend analysis of the DNA methylation level changes in four patients assessed at the time of diagnosis and in remission of the disease after chemotherapy course revealed a significant decrease of CpGs methylation in all MM patients reaching therapeutic response.Conclusions: Updated data on the epigenetic analysis in patients with monoclonal gammopathies confirmed an important role of the RIL gene in the epigenetic network acting in pathogenesis of MM. The presented results suggest possible prognostic value and therapeutic target in patients with MM. In contrast, the p15INK4b gene known as a frequent focus of epigenetic modifications in many tumors, did not show statistically significant differences in our study. Our results indicate that for DNA methylation analyses of MM or MGUS patients, the RIL gene could be more useful marker than the p15INK4b gene. Moreover, the decrease of the methylation level in patients undergoing systemic chemotherapy might be associated with treatment response, suggesting its potential prognostic value.The paper was supported by the grant IGA MZ CR NT14393. DisclosuresNo relevant conflicts of interest to declare.

Morphological changes of Paulownia seedlings infected phytoplasmas reveal the genes associated with witches' broom through AFLP and MSAP.

Paulownia witches' broom (PaWB) caused by phytoplasma might result in devastating damage to the growth and wood production of Paulownia. To study the effect of phytoplasma on DNA sequence and to discover the genes related to PaWB occurrence, DNA polymorphisms and DNA methylation levels and patterns in PaWB seedlings, the ones treated with various concentration of methyl methane sulfonate (MMS) and healthy seedlings were investigated with amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP). Our results indicated that PaWB seedlings recovered a normal morphology, similar to healthy seedlings, after treatment with more than 20 mg·L−1 MMS; Phytoplasma infection did not change the Paulownia genomic DNA sequence at AFLP level, but changed the global DNA methylation levels and patterns; Genes related to PaWB were discovered through MSAP and validated using quantitative real-time PCR (qRT-PCR). These results implied that changes of DNA methylation levels and patterns were closely related to the morphological changes of seedlings infected with phytoplasmas.

G.P.287: Genetic and epigenetic determinants of low dysferlin expression in monocytes

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle and peripheral blood monocytes. Dysferlin expression in skeletal muscle and monocytes strongly correlate in the healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While eighteen of these individuals with protein levels in the range of 40–64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of these 18 volunteers detected missense variants in only four. Analysis of DNA methylation patterns at DYSF locus showed no changes in DNA methylation levels at CpG island and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, results of the monocyte assay can not be used when determining carrier status and molecular analysis of DYSF must be performed.

Transcriptional control of fleshy fruit development and ripening.

Fleshy fruits have evolved to be attractive to frugivores in order to enhance seed dispersal, and have become an indispensable part of the human diet. Here we review the recent advances in the understanding of transcriptional regulation of fleshy fruit development and ripening with a focus on tomato. While aspects of fruit development are probably conserved throughout the angiosperms, including the model plant Arabidopsis thaliana, it is shown that the likely orthologues of Arabidopsis genes have distinct functions in fleshy fruits. The model for the study of fleshy fruit development is tomato, because of the availability of single gene mutants and transgenic knock-down lines. In other species, our knowledge is often incomplete or absent. Tomato fruit size and shape are co-determined by transcription factors acting during formation of the ovary. Other transcription factors play a role in fruit chloroplast formation, and upon ripening impact quality aspects such as secondary metabolite content. In tomato, the transcription factors NON-RIPENING (NOR), COLORLESS NON-RIPENING (CNR), and RIPENING INHIBITOR (MADS-RIN) in concert with ethylene signalling regulate ripening, possibly in response to a developmental switch. Additional components include TOMATO AGAMOUS-LIKE1 (TAGL1), APETALA2a (AP2a), and FRUITFULL (FUL1 and FUL2). The links between this highly connected regulatory network and downstream effectors modulating colour, texture, and flavour are still relatively poorly understood. Intertwined with this network is post-transcriptional regulation by fruit-expressed microRNAs targeting several of these transcription factors. This important developmental process is also governed by changes in DNA methylation levels and possibly chromatin remodelling.

Changes in DNA Methylation Levels and Nuclear Distribution Patterns after Microspore Reprogramming to Embryogenesis in Barley

Under specific stress treatments, the microspore can be induced in vitro to deviate from its gametophytic development and to reprogram towards embryogenesis, becoming a totipotent cell and forming haploid embryos. These can further regenerate homozygous plants for production of new isogenic lines, an important biotechnological tool for crop breeding. DNA methylation constitutes a prominent epigenetic modification of the chromatin fiber which regulates gene expression. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation; however, the relationship between global DNA methylation and genome-wide expression patterns is still poorly understood. In this work, the dynamics of global DNA methylation levels and distribution patterns were analyzed during microspore reprogramming to embryogenesis and during pollen development in Hordeum vulgare. Quantification of global DNA methylation levels and 5-methyl-deoxycytidine (5mdC) immunofluorescence were conducted at specific stages of pollen development and after reprogramming to embryogenesis to analyze the epigenetic changes that accompany the change of developmental program and cell fate. The results showed low DNA methylation levels in microspores and a high increase along pollen development and maturation; an intense 5mdC signal was concentrated in the generative and sperm nuclei whereas the vegetative nucleus exhibited a weaker DNA methylation signal. After inductive stress treatment, low methylation levels and faint 5mdC signals were observed in nuclei of reprogrammed microspores and 2-4-cell proembryos. This data revealed a global DNA hypomethylation during the change of the developmental program and first embryogenic divisions. This is in contrast with the hypermethylation of generative and sperm cells of the male germline during pollen maturation, suggesting an epigenetic regulation after induction of microspore embryogenesis. At later embryogenesis stages, global DNA methylation progressively increased, accompanying embryo development and differentiation events like in zygotic embryos, corroborating that DNA methylation is critical for the regulation of gene expression in microspore embryogenesis.

Characterization of DNA Methylation Variations During Fruit Development and Ripening of Sweet Orange

DNA methylation is a type of epigenetic modification involved in many biological processes. Recent studies have shown that DNA methylation plays important roles in regulating fruit development and ripening. In the present study, sweet oranges at six developmental stages were investigated to understand the possible roles of DNA methylation during fruit development and ripening. Analysis of genomic DNA methylation levels in different fruit tissues and developmental stages indicated that methylation levels were not static during fruit development and ripening. To explore the possible contributions of methylation-related genes in fruit development and ripening process, the full-length coding sequences corresponding to three types of DNA methyltransferase families (CsMET1, CsCMT3, and CsDRM1), a chromatin-remodeling gene (CsDDM1), and a demethylation gene (CsDME1) were isolated. The expression profiles of these methylation-related genes were analyzed in vegetative tissues (seedlings and leaves) and reproductive tissues (three stages of flowering and six stages of fruit development). The results showed that CsDRM1 was highly expressed in seedlings, leaves, and flowers compared with other genes. During fruit development and ripening, CsMET1, CsCMT1, CsCMT2, CsDRM1, and CsDMEs showed higher expression in the peel than in the flesh. However, global DNA methylation level changes could not be explained by the expression of a single gene, suggesting that the methylation and demethylation system may cooperatively contribute to the final global DNA methylation pattern.

Detection of genome DNA methylation change in spinach induced by 5-azaC

DNA methylation has been implicated in the regulation of gene expression, genome imprinting, and chromatin remodeling in eukaryotes. In this study, we analyzed possible alterations in levels and patterns of cytosine methylation in male and female spinach plants after treatment with demethylation agent 5-azacytidine (5-azaC) using two methods: (1) direct determination of 5-methylcytidine (5 mC) amounts in genomic DNA by high-performance liquid chromatography (HPLC) separation and quantification of nucleosides and (2) methylation-sensitive inter-simple sequence repeat (MS-ISSR) technique. HPLC analysis revealed that the DNA methylation events in male and female spinach leaves markedly decreased upon 30 μM 5-azaC treatment, and the methylation level gradually decreased with the increase in 5-azaC concentration. To study the altered DNA methylation patterns in spinach after 5-azaC treatment, untreated and 500 μM 5-azaC-treated samples were analyzed by MS-ISSR assay. A total of 385 informative profiles were resolved using 35 ISSR primer sets. MS-ISSR analysis showed various altered methylation patterns between untreated and 5-azaC-treated spinach plants. These alterations were mainly demethylation events, which were largely consistent with the HPLC results. Both HPLC and MS-ISSR analyses showed that the changes in DNA methylation levels and patterns were similar in male and female spinach leaves, which implies that sex was not the main factor influencing DNA methylation levels and patterns in the vegetative organs of spinach. This study could provide a molecular basis of the altered DNA methylation induced by 5-azaC, and lay a foundation for further investigation of the relationship between methylation and sex determination and development in this dioecious plant spinach.

Genome-Wide DNA Methylation Analysis of Systemic Lupus Erythematosus Reveals Persistent Hypomethylation of Interferon Genes and Compositional Changes to CD4+ T-cell Populations

Systemic lupus erythematosus (SLE) is an autoimmune disease with known genetic, epigenetic, and environmental risk factors. To assess the role of DNA methylation in SLE, we collected CD4+ T-cells, CD19+ B-cells, and CD14+ monocytes from 49 SLE patients and 58 controls, and performed genome-wide DNA methylation analysis with Illumina Methylation450 microarrays. We identified 166 CpGs in B-cells, 97 CpGs in monocytes, and 1,033 CpGs in T-cells with highly significant changes in DNA methylation levels (p<1×10−8) among SLE patients. Common to all three cell-types were widespread and severe hypomethylation events near genes involved in interferon signaling (type I). These interferon-related changes were apparent in patients collected during active and quiescent stages of the disease, suggesting that epigenetically-mediated hypersensitivity to interferon persists beyond acute stages of the disease and is independent of circulating interferon levels. This interferon hypersensitivity was apparent in memory, naïve and regulatory T-cells, suggesting that this epigenetic state in lupus patients is established in progenitor cell populations. We also identified a widespread, but lower amplitude shift in methylation in CD4+ T-cells (>16,000 CpGs at FDR<1%) near genes involved in cell division and MAPK signaling. These cell type-specific effects are consistent with disease-specific changes in the composition of the CD4+ population and suggest that shifts in the proportion of CD4+ subtypes can be monitored at CpGs with subtype-specific DNA methylation patterns.

Infection with influenza A viruses causes changes in promoter DNA methylation of inflammatory genes

Replication of influenza virus in the host cells results in production of immune mediators like cytokines. Excessive secretion of cytokines (hypercytokinemia) has been observed during highly pathogenic avian influenza virus (HPAI-H5N1) infections resulting in high fatality rates. The exact mechanism of hypercytokinemia during influenza virus infection is still not known completely. As promoter DNA methylation changes are linked with expression changes in genes, we intend to identify whether changes in promoter DNA methylation have any role in expression of cytokines during influenza A virus infection. A panel of 24 cytokine genes and genes known to be involved in inflammatory response were analyzed for their promoter DNA methylation changes during influenza A virus infections. Four different strains of influenza A viruses, viz. H5N1, H1N1, pandemic (2009) H1N1, and a vaccine strain of H5N1, were used for the study. We found seven of the total 24 inflammatory genes studied, showing significant changes in their promoter methylation levels in response to virus infection. These genes included proinflammatory cytokines CXCL14, CCL25, CXCL6, and interleukines IL13, IL17C, IL4R. The changes in DNA methylation levels varied across different strains of influenza viruses depending upon their virulence. Significant promoter hypomethylation in IL17C and IL13 genes was observed in cells infected with HPAI-H5N1 virus compared with other influenza viruses. This decrease in methylation was found to be positively correlating with the increased expression of these genes. Analysis of IL17C promoter region using bisulfite sequencing resulted in identification of a CpG site within Retinoid X receptor-alpha (RXR-α) transcription factor binding site undergoing demethylation specifically in H5N1-infected cells but not in other influenza-infected cells. Thus, the study could demonstrate that changes in promoter methylation in certain specific cytokine genes actually have a possible role in their expression changes during influenza A virus infection.