Comparative WGBS identifies genes that influence non-ripe phenotype in tomato epimutant <Emphasis Type="Italic">Colourless non-ripening</Emphasis>

Whole-genome bisulfite sequencing (WGBS) allows single-base resolution and genome-wide profiling of DNA methylation in plants and animals. This technology provides a powerful tool to identify genes that are potentially controlled by dynamic changes of DNA methylation and demethylation. However, naturally occurring epimutants are rare and genes under epigenetic regulation as well as their biological relevances are often difficult to define. In tomato, fruit development and ripening are a complex process that involves epigenetic control. We have taken the advantage of the tomato epimutant Colourless non-ripening (Cnr) and performed comparative mining of the WGBS datasets for the Cnr and SlCMT3-silenced Cnr fruits. We compared DNA methylation profiles for the promoter sequences of approximately 5,000 bp immediately upstream of the coding region of a list of 20 genes. Differentially methylated regions were found for some of these genes. Virus-induced gene silencing (VIGS) of differentially methylated gene SlDET1 or SlPDS resulted in unusual brown pigmentation in Cnr fruits. These results suggest that comparative WGBS coupled with VIGS can be used to identify genes that may contribute to the colourless unripe phenotype of fruit in the Cnr epimutant.     

Novel markers of gene methylation and expression in breast cancer

An optimized methylation-sensitive restriction fingerprinting technique was used to search for differentially methylated CpG islands in the tumor genome and detected seven genes subject to abnormal epigenetic regulation in breast cancer: SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1. For each gene, the rate of promoter methylation and changes in expression were estimated in tumor and morphologically intact paired specimens of breast tissue (N = 100). Significant methylation rates of 38, 18, and 8% were found for SEMA6B, BIN1, and LAMC3, respectively. The genes were not methylated in morphologically intact breast tissue. The expression of SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1 was decreased in 44–94% of tumor specimens by the real-time RT-PCR assay. The most profound changes in SEMA6B and LAMC3 suggest that these genes can be included in biomarker panels for breast cancer diagnosis. Fine methylation mapping of the most frequently methylated CpG islands (SEMA6B, BIN1, and LAMC3) provides a fundamental basis for developing efficient methylation tests for these genes.     

Plant DNA methyltransferase genes: Multiplicity,expression, methylation patterns

Expression and methylation patterns of genes encoding DNA methyltransferases and their functionally related proteins were studied in organs of Arabidopsis thaliana plants. Genes coding for the major maintenance-type DNA methyltransferases, MET1 and CMT3, and the major de novo-type DNA methyltransferase, DRM2, are actively expressed in all organs. Similar constitutively active expression was observed for genes encoding their functionally related proteins, a histone H3K9 methyltransferase KYP and a catalytically non-active protein DRM3. Expression of the MET1 and CMT3 genes is significantly lower in developing endosperm compared with embryo. Vice versa, expression of the MET2a, MET2b, MET3, and CMT2 genes in endosperm is much more active compared with embryo. A special maintenance DNA methylation system seems to operate in endosperm. The DNMT2 and N6AMT genes encoding putative methyltransferases are constitutively expressed at low levels. CMT1 and DRM1 genes are expressed rather weakly in all investigated organs. Most of the studied genes have methylation patterns conforming to the “body-methylated gene” prototype. A peculiar feature of the MET family genes is methylation at all three possible site types (CG, CHG, and CHH). The most weakly expressed among genes of their respective families, CMT1 and DRM1, are practically unmethylated. The MET3 and N6AMT genes have unusual methylation patterns, promoter region, and most of the gene body devoid of any methylation, and the 3'-end proximal part of the gene body is highly methylated.     

Genome-wide mapping of DNA methylation in Nile Tilapia

Cytosine DNA methylation is crucial for gene regulation and maintenance of genome stability. However, the detailed nile tilapia methylome remains uncharacterized. In this study, we present the first high-resolution methylome of tilapia gonad generated using methylated DNA immunoprecipitation (MeDIP) and high-throughput sequencing. In the ovary, 265 and 56 methylation peaks were identified in the genebody and promoter region of 145 genes, respectively. In the testis, 293 and 80 methylation peaks were identified in the genebody and promoter region of 144 genes. Furthermore, 8 and 49 genes showed differentially higher and lower promoter-region methylation rates, respectively, in the ovary relative to those of the testis. Quantitative PCR results revealed that the expression level of fibroblast growth factor 16 (fgf16), sialidase-3-like, fibroblast growth factor 20, aromatase (cyp19a), estrogen receptor, and gonadotropin receptor II precursor were negatively correlated to their methylation levels in the ovary and testis. The methylated levels of cyp19a and fgf16 were validated by bisulfite sequencing PCR technology, and the results were consistent with the MeDIP results. Thus, apart from generating the first methylation map, this study produced a candidate gene repository that provides additional options to explore the relationship between DNA methylation and sex differentiation or maintenance.     

Functional Analyses of <Emphasis Type="Italic">PtROS1</Emphasis>-RNAi in Poplars and Evaluation of Its Effect on DNA Methylation

DNA methylation occurs mostly at the C5 position of dinucleotide symmetric CpG sites in genomic DNA. A balance is maintained in the plant genome between DNA methylation mediated by RNA-directed DNA methylation (RdDM) and DNA demethylation mediated by the DEMETER (DME) protein family and REPRESSOR OF SILENCING (ROS1). We used double-stranded RNA (dsRNA) silencing to suppress ROS1 protein expression in ‘Nanlin895’ (Populus deltoides × Populus euramericana ‘Nanlin895’). Leaves of WT and transformant poplars revealed more symmetric methylation on CpG sites than roots and stems. In addition, leaves of transformant poplars revealed more methylated CpG sites in both 5.8S rDNA and histone H3 compared to WT types via 0, 50 and 100 mM NaCl treatments. In asymmetric methylation sites, transformant poplars exhibited more methylated CpHpG and CpHpH contexts than WT poplars. On the other hand, hypermethylation induced by PtROS1-RNAi construct resulted in pleiotropic phenotypic changes in transgenic poplars. The percentage of wavy leaves was increased maximum by ~45% in transgenic poplars. Also, the number of leaves was increased by ~200 number in transformants. Furthermore, shooting (%) and rooting (%) was decreased in transgenic poplars versus WT.     

Comparison of novel and existing methods for detecting differentially methylated regions

Background

Single-probe analyses in epigenome-wide association studies (EWAS) have identified associations between DNA methylation and many phenotypes, but do not take into account information from neighboring probes. Methods to detect differentially methylated regions (DMRs) (clusters of neighboring probes associated with a phenotype) may provide more power to detect associations between DNA methylation and diseases or phenotypes of interest.

Results

We proposed a novel approach, GlobalP, and perform comparisons with 3 methods—DMRcate, Bumphunter, and comb-p—to identify DMRs associated with log triglycerides (TGs) in real GAW20 data before and after fenofibrate treatment. We applied these methods to the summary statistics from an EWAS performed on the methylation data. Comb-p, DMRcate, and GlobalP detected very similar DMRs near the gene CPT1A on chromosome 11 in both the pre- and posttreatment data. In addition, GlobalP detected 2 DMRs before fenofibrate treatment in the genes ETV6 and ABCG1. Bumphunter identified several DMRs on chromosomes 1 and 20, which did not overlap with DMRs detected by other methods.

Conclusions

Our novel method detected the same DMR identified by two existing methods and detected two additional DMRs not identified by any of the existing methods we compared.

     

Genome-wide DNA methylation profiles of maternal peripheral blood and placentas: potential risk factors for preeclampsia and validation of <Emphasis Type="Italic">GRK5</Emphasis>

Hypoxic placentation has been considered as a key step for the development of preeclampsia (PE); however, the underlying epigenetic mechanisms are still not fully understood. The purpose of this study is to investigate the whole genome DNA methylation status of PE. A microarray analysis using the Infinium HumanMethylation450 BeadChip assay in the placentas and maternal peripheral blood (PB) from PE patients and normal controls was performed. For validation, a quantitative RT-PCR analysis was used. Maternal PB showed 71 differentially methylated CpG loci (44 hypermethylated and 27 hypomethylated), while placenta revealed 365 loci (37 hypermethylated and 328 hypomethylated) at the statistical significance level of |Δβ| ≥ 0.17 and P ≤ 0.01. Notably, among the candidates showing significant signals, GRK5 (a member of G protein-coupled receptor kinase family that has previously been known to be associated with PE) showed a significantly hypomethylated level in the placentas of PE patients (Δβ = ?0.176, P = 2.8 × 10^?5). In the validation for the potential effect of GRK5 methylation on the gene regulation, GRK5 expression was significantly increased in the placentas from PE patients compared to those from controls (P = 0.027). In further GO analysis, genes of MHC class II protein complex showed the most significant differential methylation in the maternal PB of PE patients, while genes of palate development were differentially methylated in the placenta. Although further replication and functional studies are required, our preliminary results suggest that PE has distinct DNA methylation profiles in the maternal PB and placentas, which may provide insight into future research.     

Dynamics of LINE-1 retrotransposon methylation levels in circulating DNA from lung cancer patients undergoing antitumor therapy

Malignant cell transformation is accompanied with abnormal DNA methylation, such as the hypermethylation of certain gene promoters and hypomethylation of retrotransposons. In particular, the hypomethylation of the human-specific family of LINE-1 retrotransposons was observed in lung cancer tissues. It is also known that the circulating DNA (cirDNA) of blood plasma and cell-surface-bound circulating DNA (csb-cirDNA) of cancer patients accumulate tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. This work compares LINE-1 retrotransposon methylation patterns in cirDNA of 16 lung cancer patients before and after treatment. CirDNA was isolated from blood plasma, and csb-cirDNA fractions were obtained by successive elution with EDTA-containing phosphate buffered saline and trypsin. Concentrations of methylated LINE-1 region 1 copies (LINE-1-met) were assayed by real-time methylation-specific PCR. LINE-1 methylation levels were normalized to the concentration of LINE-1 region 2, which was independent of the methylation status (LINE-1-Ind). The concentrations of LINE-1-met and LINE-1-Ind in csb-cirDNA of lung cancer patients exhibited correlations before treatment (r = 0.54), after chemotherapy (r = 0.72), and after surgery (r = 0.83) (P < 0.05, Spearman rank test). In the total group of patients, the level of LINE-1 methylation (determined as the LINE-1-met/LINE-1-Ind ratio) was shown to increase significantly during the follow-up after chemotherapy (P < 0.05, paired t test) and after surgery compared to the level of methylation before treatment (P < 0.05, paired t test). The revealed association between the level of LINE-1 methylation and the effect of antitumor therapy was more pronounced in squamous cell lung cancer than in adenocarcinoma (P < 0.05 and P > 0.05, respectively). These results suggest a need for the further investigation of dynamic changes in levels of LINE-1 methylation depending on the antitumor therapy.