Exploring disease-specific methylated CpGs in human male genital abnormalities by using methylated-site display-amplified fragment length polymorphism (MSD-AFLP).

The incidence of male reproductive system disorders, especially hypospadias, has been increasing in developed countries since the latter half of the 20th century. Endocrine-disrupting chemicals from the environment are considered to be involved in hypospadias onset through epigenetic alterations. This pilot study aimed to explore disease-specific methylated CpGs in human patient samples using the methylated-site display-amplified fragment length polymorphism (MSD-AFLP) technique developed by our research group [1]. We compared clinical samples from hypospadias and phimosis patients. Foreskin and blood samples were collected from one- to two-year-old patients with hypospadias (N = 3) and phimosis (N = 3) during surgical treatment. MSD-AFLP analysis showed significantly decreased CpG-methylation levels of genes such as MYH11 and increased CpG-methylation levels of genes such as PLA2G15 in hypospadias patients. Hierarchical clustering analysis showed that genes with significantly altered CpG levels were more markedly altered in DNA from blood than from foreskin. Because of the small number of samples, further investigation is necessary to elucidate the association between variations in CpG levels in foreskin and blood DNA and male genital abnormalities. However, our MSD-AFLP method appears to be a useful tool for exploring disease-specific methylated-CpGs in human epidemiological studies.

Does the prenatal bisphenol A exposure alter DNA methylation levels in the mouse hippocampus?: An analysis using a high-sensitivity methylome technique.

BACKGROUND: There is still considerable debate about the effects of exposure to bisphenol A (BPA) an endocrine disrupter at low doses. Recently, many studies using animal models have shown that prenatal BPA exposure induces behavioral and neuronal disorders due to epigenetic changes in the brain. However, striking evidence of epigenomic changes has to be shown. METHODS: To investigate whether low-dose BPA exposure in the fetal stage can alter CpG methylation levels in the central nervous system, the hippocampus of the inbred C57BL/6 J mouse as the target tissue was collected to detect alterations in CpG methylation levels using a highly sensitive method of genome-wide DNA methylation analysis, methylated site display-amplified fragment length polymorphism (MSD-AFLP). RESULTS: BPA showed the sex-hormone like effects on male reproductive organs. Although we examined the methylation levels of 43,840 CpG sites in the control and BPA (200 µg/kg/day)-treated group (6 mice per group), we found no statistically significant changes in methylation levels in any CpG sites. CONCLUSIONS: At least under the experimental condition in this study, it is considered that the effect of low-dose BPA exposure during the fetal stage on hippocampal DNA methylation levels is extremely small.

Efficient identification of NLR by using a genome-wide protein domain and motif survey program, Ex-DOMAIN.

Genomic and amino acid sequences of organisms are freely available from various public databases. We designed a genome-wide survey program, named "Ex-DOMAIN" (exhaustive domain and motif annotator using InterProScan), of protein domains and motifs to aid in the identification and characterization of proteins by using the InterProScan sequence analysis application, which can display information and annotations of targeted proteins and genes, conserved protein domains and motifs, chromosomal locations, and structural diversities of target proteins. In this study, we indicated the disease resistance genes (proteins) that play an important role in defense against pathogens in Arabidopsis thaliana (thale cress) and Cucumis sativus (cucumber), by searches based on the conserved protein domains, NB-ARC (a nucleotide-binding adaptor shared by the apoptotic protease-activating factor-1, plant resistance proteins, and Caenorhabditis elegans death-4 protein) and C-terminal leucine-rich repeat (LRR), in the nucleotide-binding domain and LRR (NLR) proteins. Our findings suggest that this program will enable searches for various protein domains and motifs in all organisms.

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

BACKGROUND: It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. RESULTS: Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. CONCLUSION: MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

High-coverage gene expression profiling analysis of the cellulase-producing fungus Acremonium cellulolyticus cultured using different carbon sources.

The gene expression of a cellulase-producing fungus, Acremonium cellulolyticus, was investigated after culturing with three different carbon sources: glycerol, lactose, and Solka-Floc powdered cellulose (SF). High-coverage gene expression profiling (HiCEP) analysis, a method requiring no prior sequence knowledge, was used to screen genes upregulated at the early stage of cellulase production. SF was used as a strong inducer of cellulase production, lactose was used as an inducer of the expression of cellulase genes at the early stage of the culture, and glycerol was used as a negative control. Approximately 15,000 transcript-derived fragments (TDFs) were detected in each sample prepared from the culture grown for 16 h. Based on the expression profiles of the cultured cells, 36 fragments upregulated in both the SF and lactose cultures were selected and sequenced. The deduced gene products of 31 TDFs were likely related to biomass degradation, sugar metabolism, transcriptional regulation, protein modification and metabolism, cell wall recycling, fatty acid and polyketide biosynthesis, and other functions. Quantitative real-time reverse-transcriptase polymerase chain reaction analysis verified that almost all of the transcripts obtained by HiCEP analysis were upregulated in the SF and lactose cultures grown for 18 h. Some of the TDFs in the SF culture were further upregulated over the course of 72 h. The gene products from these TDFs would provide insight into improving the cellulase productivity of A. cellulolyticus.

A comprehensive analysis of the soybean genes and proteins expressed under flooding stress using transcriptome and proteome techniques.

The inducible genes and proteins were analyzed using transcriptome and proteome techniques to explore the mechanisms underlying soybean response to flooding stress. Soybean seedlings were germinated for 2 days and subjected to flooding for 12 h, and the total RNAs and proteins were extracted from the root and hypocotyl. High-coverage gene expression profiling analysis as transcriptome technique was performed. Ninety-seven out of the 29,388 peaks observed demonstrated a greater than 25-fold change following 12 h of flood-induced stress. Furthermore, 34 proteins out of 799 proteins were changed by 12 h stress. Genes associated with alcohol fermentation, ethylene biosynthesis, pathogen defense, and cell wall loosening were significantly up-regulated. Hemoglobin, acid phosphatase, and Kunitz trypsin protease inhibitor were altered at both transcriptional and translational levels. Reactive oxygen species scavengers and chaperons were changed only at the translational level. It is suggested that the early response of soybean under flooding might be important stress adaptation to ensure survival against not only hypoxia but also the direct damage of cell by water.

Soybean proteome database: a data resource for plant differential omics.

The Soybean Proteome Database aims to be a data repository for functional analyses of soybean responses to flooding injury, recognized as a major constraint for establishment and production of this plant. The current release contains 21 reference maps of soybean (Glycine max cv. Enrei) proteins electrophoresed on two-dimensional polyacrylamide gels of which the samples were collected from several organs, tissues and organelles. These reference maps include 7311 detected proteins and 532 identified proteins, or proteins for which a sequence or peptide peak has been determined. The database is searchable by protein properties such as accession number, description and isoelectric point and molecular weight range. The Soybean Proteome Database also integrates multiple "omes". An omics table reveals relationships among 106 mRNAs, 51 proteins and 89 metabolites that vary over time under flooding stress. The tabulated metabolites are anchored to a metabolome network. A unified temporal-profile tag attached to the mRNAs, proteins and metabolites facilitates retrieval of the data based on the temporal expression profiles. A graphical user interface based on dynamic HTML facilitates viewing the metabolome network as well as the profiles of multiple omes in a uniform manner. The entire database is available at http://proteome.dc.affrc.go.jp/Soybean/.