Ultra-deep whole genome bisulfite sequencing reveals a single methylation hotspot in human brain mitochondrial DNA

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Bisulfite sequencing Data Presentation and Compilation (BDPC) web server--a useful tool for DNA methylation analysis

During bisulfite genomic sequencing projects large amount of data are generated. The Bisulfite sequencing Data Presentation and Compilation (BDPC) web interface (http://biochem.jacobs-university.de/BDPC/) automatically analyzes bisulfite datasets prepared using the BiQ Analyzer. BDPC provides the following output: (i) MS-Excel compatible files compiling for each PCR product (a) the average methylation level, the number of clones analyzed and the percentage of CG sites analyzed (which is an indicator of data quality), (b) the methylation level observed at each CG site and (c) the methylation level of each clone. (ii) A methylation overview table compiling the methylation of all amplicons in all tissues. (iii) Publication grade figures in PNG format showing the methylation pattern for each PCR product embedded in an HMTL file summarizing the methylation data, the DNA sequence and some basic statistics. (iv) A summary file compiling the methylation pattern of different tissues, which is linked to the individual HTML result files, and can be directly used for presentation of the data in the internet. (v) A condensed file, containing all primary data in simplified format for further downstream data analysis and (vi) a custom track file for display of the results in the UCSC genome browser.     

DNA from Aspergillus flavus contains 5-methylcytosine

DNA from Aspergillus sp. has been reported not to contain 5-methylcytosine. However, it has been found that Aspergillus nidulans responds to 5-azacytidine, a drug that is a strong inhibitor of DNA methyltransferases. Therefore, we have re-examined the occurrence of 5-methylcytosine in DNA from Aspergillus flavus by using a highly sensitive and specific method for detection of modified bases in genomic DNA comprising high-performance liquid chromatography separation of nucleosides, labeling of the nucleoside with deoxynucleoside kinase and two-dimensional thin-layer chromatography. Our results show that 5-methylcytosine is present in DNA from A. flavus. We estimate the relative amounts of 5-methylcytosine to cytosine to be approximately 1/400.     

Bisulphite sequencing reveals dynamic DNA methylation under desiccation and salinity stresses in rice cultivars

DNA methylation governs gene regulation in plants in response to environmental conditions. Here, we analyzed role of DNA methylation under desiccation and salinity stresses in three (IR64, stress-sensitive; Nagina 22, drought-tolerant and Pokkali, salinity-tolerant) rice cultivars via bisulphite sequencing. Methylation in CG context within gene body and methylation in CHH context in distal promoter regions were positively correlated with gene expression. Hypomethylation in Nagina 22 and hypermethylation in Pokkali in response to desiccation and salinity stresses, respectively, were correlated with higher expression of few abiotic stress response related genes. Most of the differentially methylated and differentially expressed genes (DMR-DEGs) were cultivar-specific, suggesting an important role of DNA methylation in abiotic stress responses in rice in cultivar-specific manner. DMR-DEGs harboring differentially methylated cytosines due to DNA polymorphisms between the sensitive and tolerant cultivars in their promoter regions and/or coding regions were identified, suggesting the role of epialleles in abiotic stress responses.     

Targeted methylation sequencing reveals dysregulated Wnt signaling in Parkinson disease

Parkinson disease(PD) is a progressive neurodegenerative movement disorder. Both environmental and genetic factors play important roles in PD etiology. A number of environmental toxins cause parkinsonism in human and animal models. Genetic studies of rare early onset familial PD cases resulted in identification of disease-linked mutations in multiple genes. Nevertheless, the potential interaction between environment and genetics in PD pathogenesis remains largely unknown. We hypothesized that environmental factors induce abnormal epigenetic regulation that is involved in the pathogenesis of both familial and sporadic PD. We determined the global methylation status of 80,000e110,000 Cp G sites in each of the five sporadic PD patient brains and five age and postmodern interval matched control brains utilizing bisulfite padlock sequencing. Multiple genes involved in neurogenesis, particularly the ones in the Wnt signaling pathway, were hypermethylated in PD brains compared to their matched control brains. Consistent with the DNA methylation changes, marked reduction of protein expression was observed for four Wnt and neurogenesis related genes(FOXC1, NEURG2, SPRY1, and CTNNB1) in midbrain dopaminergic(DA) neurons of PD. The treatment of low concentration of 1-methyl-4-phenylpyridinium(MPPt) for cells resulted in downregulation of Wnt related genes. The study revealed an important link between the epigenetic disregulation of Wnt signaling and the pathogenesis and progression of PD.     

Genome-wide analysis reveals DNA methylation markers that vary with both age and obesity

The combination of the obesity epidemic and an aging population presents growing challenges for the healthcare system. Obesity and aging are major risk factors for a diverse number of diseases and it is of importance to understand their interaction and the underlying molecular mechanisms. Herein the authors examined the methylation levels of 27578 CpG sites in 46 samples from adult peripheral blood. The effect of obesity and aging was ascertained with general linear models. More than one hundred probes were correlated to aging, nine of which belonged to the KEGG group map04080. Additionally, 10 CpG sites had diverse methylation profiles in obese and lean individuals, one of which was the telomerase catalytic subunit (TERT). In eight of ten cases the methylation change was reverted between obese and lean individuals. One region proved to be differentially methylated with obesity (LINC00304) independent of age. This study provides evidence that obesity influences age driven epigenetic changes, which provides a molecular link between aging and obesity. This link and the identified markers may prove to be valuable biomarkers for the understanding of the molecular basis of aging, obesity and associated diseases.     

Semiconductor-based sequencing of genome-wide DNA methylation states

Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we developed a MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is low cost, rapid, and scalable. We applied this protocol to demonstrate MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450 BeadChip array, and accurately identifies sites of differential DNA methylation. Furthermore, we applied an integrative approach to further investigate and confirm the role of DNA methylation in alternative splicing and to profile 5mC and 5hmC variants of DNA methylation in normal human brain tissue that is localized over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions.     

Global analysis of DNA methylation by Methyl-Capture sequencing reveals epigenetic control of cisplatin resistance in ovarian cancer cell

Cisplatin resistance is one of the major reasons leading to the high death rate of ovarian cancer. Methyl-Capture sequencing (MethylCap-seq), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein with NGS, global and unbiased analysis of global DNA methylation patterns. We applied MethylCap-seq to analyze genome-wide DNA methylation profile of cisplatin sensitive ovarian cancer cell line A2780 and its isogenic derivative resistant line A2780CP. We obtained 21,763,035 raw reads for the drug resistant cell line A2780CP and 18,821,061reads for the sensitive cell line A2780. We identified 1224 hyper-methylated and 1216 hypomethylated DMRs (differentially methylated region) in A2780CP compared to A2780. Our MethylCap-seq data on this ovarian cancer cisplatin resistant model provided a good resource for the research community. We also found that A2780CP, compared to A2780, has lower observed to expected methylated CpG ratios, suggesting a lower global CpG methylation in A2780CP cells. Methylation specific PCR and bisulfite sequencing confirmed hypermethylation of PTK6, PRKCE and BCL2L1 in A2780 compared with A2780CP. Furthermore, treatment with the demethylation reagent 5-aza-dC in A2780 cells demethylated the promoters and restored the expression of PTK6, PRKCE and BCL2L1.     

Genetic characterization of Brazilian strains of Aspergillus flavus using DNA markers

The Aspergillus genus belongs to a filamentous fungal group characterized by wide dispersion in the environment. Some species are associated with diseases, especially in immunocompromised patients, while others are of economical importance due to aflatoxin production or biotechnological applications. Its species identification is nowadays performed by traditional techniques combined with molecular markers, resulting in a higher efficiency of isolate characterization. In the present study, internal transcribed spacer, inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of Aspergillus flavus and strains of other species of the A. flavus group. High genetic diversity was revealed by RAPD and by ISSR, in which the use of the (GACA)4 primer yielded a higher diversity than with the (GTG)5 primer, although the latter showed a characteristic banding profile for each species. These data were used to create a similarity matrix for the construction of dendrograms by means of the UPGMA method. The ISSR and RAPD profiles showed that among the strains previously identificated as A. flavus, one should be A. oryzae, one A. parasiticus and two A. tamarii. On the other hand, a strain previously identified as A. parasiticus should be A. flavus. All these strains were retested by traditional methods and their new species identification was confirmed. These results strongly support the need for using molecular markers as an auxiliary tool in differentiating fungal species and strains.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.     

Aspergillus nomius,a new aflatoxin-producing species related to Aspergillus flavus and Aspergillus tamarii

Aspergillus nomius is described and represents a new aflatoxigenic species phenotypically similar to A. flavus. Strains examined were isolated from insects and agricultural commodities. Separation from A. flavus is based on the presence of indeterminate sclerotia and a lower growth temperature. Comparisons of DNA relatedness show A. nomius to have only relatively recently evolved from A. flavus and A. tamarii.     

Proteomic analysis reveals an aflatoxin-triggered immune response in cotyledons of Arachis hypogaea infected with Aspergillus flavus

An immune response is triggered in host cells when host receptors recognize conserved molecular motifs, pathogen-associated molecular patterns (PAMPs), such as β-glucans, and chitin at the cell surface of a pathogen. Effector-triggered immunity occurs when pathogens deliver effectors into the host cell to suppress the first immune signaling. Using a differential proteomic approach, we identified an array of proteins responding to aflatoxins in cotyledons of peanut (Arachis hypogaea) infected with aflatoxin-producing (toxigenic) but not nonaflatoxin-producing (atoxigenic) strains of Aspergillus flavus. These proteins are involved in immune signaling and PAMP perception, DNA and RNA stabilization, induction of defense, innate immunity, hypersensitive response, biosynthesis of phytoalexins, cell wall responses, peptidoglycan assembly, penetration resistance, condensed tannin synthesis, detoxification, and metabolic regulation. Gene expression analysis confirmed the differential abundance of proteins in peanut cotyledons supplemented with aflatoxins, with or without infection with the atoxigenic strain. Similarly, peanut germination and A. flavus growth were altered in response to aflatoxin B1. These findings show an additional immunity initiated by aflatoxins. With the PAMP- and effector-triggered immune responses, this immunity constitutes the third immune response of the immune system in peanut cotyledon cells. The system is also a three-grade coevolution of plant-pathogen interaction.     

Global analysis of DNA methylation in hepatocellular carcinoma via a whole-genome bisulfite sequencing approach

Alterations in DNA methylation patterns are considered early events in hepatocellular carcinoma (HCC). However, their mechanism and significance remain to be elucidated. We studied the genome-wide DNA methylation landscape of HCC by applying whole-genome bisulfite sequencing (WGBS) techonlogy. Overall, HCC exhibits a genome-wide hypomethylation pattern. After further annotation, we obtained 590 differentially hypermethylated genes (hyper-DMGs) and 977 differentially hypomethylated genes (hypo-DMGs) from three groups. Hyper-DMGs were mainly involved in ascorbate and alternate metabolism pathways, while hypo-DMGs were mainly involved in focal adhesion. By integrating the DMGs with HCC-related differentially expressed genes (DEGs) and DMGs from the TCGA database, we constructed prognostic model based on thirteen aberrantly methylated DEGs, and verified our prognostic model in GSE14520 dataset. This study compares the patterns of global epigenomic DNA methylation during the development of HCC, focusing on the role of DNA methylation in the early occurrence and development of HCC, providing a direction for future research on its epigenetic mechanism.     

Lack of Host Specialization in Aspergillus flavus

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22°C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton bolls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.     

Lack of host specialization in Aspergillus flavus

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22 degrees C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton balls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.