Analysis of soybean DNA methylomes shows CHH hypermethylation enhances gene expression in cotyledons of developing seeds [mRNA]
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ABSTRACT: Cytosine methylation is an important mechanism for dynamical regulation of gene expression and transposon mobility during plant developmental processes. Recently, the variation of DNA methylation has been described between wild type and DNA methylation-related mutants in Arabidopsis thaliana. However, the elaborate representation of soybean DNA methylomes remains lacking. Here, we described the epigenome maps of soybean root, stem, leaf, and cotyledon of developing seed at a single-base resolution. We confirmed the transcription start sites of genes using high-throughput sequencing and reported the DNA methylation patterns in gene and transposon regions. The correlation between gene expression and DNA methylation was revealed through transcriptome sequencing. We found CHH methylation may function in promotion of gene expression and ten cotyledon-preferred genes were identified CHH hypermethylated in cotyledon. Small RNA library sequencing showed that DNA methylation was enhanced by small RNAs not by strand-specific way, and the variation of DNA methylation between the organs was highly related with expression of small RNAs. mRNA-Seq of roots, stems, leaves, and cotyledons of developing seeds
Project description:Cytosine methylation is an important mechanism for dynamical regulation of gene expression and transposon mobility during plant developmental processes. Recently, the variation of DNA methylation has been described between wild type and DNA methylation-related mutants in Arabidopsis thaliana. However, the elaborate representation of soybean DNA methylomes remains lacking. Here, we described the epigenome maps of soybean root, stem, leaf, and cotyledon of developing seed at a single-base resolution. We confirmed the transcription start sites of genes using high-throughput sequencing and reported the DNA methylation patterns in gene and transposon regions. The correlation between gene expression and DNA methylation was revealed through transcriptome sequencing. We found CHH methylation may function in promotion of gene expression and ten cotyledon-preferred genes were identified CHH hypermethylated in cotyledon. Small RNA library sequencing showed that DNA methylation was enhanced by small RNAs not by strand-specific way, and the variation of DNA methylation between the organs was highly related with expression of small RNAs. methylomes of roots, stems, leaves, and cotyledons of developing seeds
Project description:Cytosine methylation is an important mechanism for dynamical regulation of gene expression and transposon mobility during plant developmental processes. Recently, the variation of DNA methylation has been described between wild type and DNA methylation-related mutants in Arabidopsis thaliana. However, the elaborate representation of soybean DNA methylomes remains lacking. Here, we described the epigenome maps of soybean root, stem, leaf, and cotyledon of developing seed at a single-base resolution. We confirmed the transcription start sites of genes using high-throughput sequencing and reported the DNA methylation patterns in gene and transposon regions. The correlation between gene expression and DNA methylation was revealed through transcriptome sequencing. We found CHH methylation may function in promotion of gene expression and ten cotyledon-preferred genes were identified CHH hypermethylated in cotyledon. Small RNA library sequencing showed that DNA methylation was enhanced by small RNAs not by strand-specific way, and the variation of DNA methylation between the organs was highly related with expression of small RNAs. small RNA profiling of roots, stems, leaves, and cotyledons of developing seeds
Project description:Proteomics on B. thuringiensis CT_43 cells in GYS medium. Two biological replicate cell samples were collected at time points of 7 h, 9 h, 13 h and 22 h, respectively. The crude proteins were purified using the ReadyPrep 2-D Cleanup Kit, underwent the reductive alkylation, tryptically digested, and were labeled with 8-plex iTRAQ reagents as follows: 7 h-1, 113; 7 h-2, 114; 9 h-1, 115; 9 h-2, 116; 13 h-1, 117; 13 h-2, 118; 22 h-1, 119; and 22 h-2, 121. The labeled samples were pooled and resolved into 12 fractions, which were loaded onto LC-MSMS.
Project description:DNA methylation stabilizes developmentally programmed gene expression states. Aberrant methylation is associated with disease progression and is a common feature of cancer genomes. Presently, few methods enable quantitative, large-scale, single-base resolution mapping of DNA methylation states in desired regions of a complex mammalian genome. Here, we present an approach that combines array-based hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in genomic regions spanning hundreds of thousands of bases. This single molecule strategy enables methylation variable positions to be quantitatively examined with high sampling precision. Using bisulfite capture, we assessed methylation patterns across 324 randomly selected CpG islands (CGI) representing more than 25,000 CpG sites. A single lane of Illumina sequencing permitted methylation states to be definitively called for >90% of target sties. The accuracy of the hybrid-selection approach was verified using conventional bisulfite capillary sequencing of cloned PCR products amplified from a subset of the selected regions. This confirmed that even partially methylated states could be successfully called. A comparison of human primary and cancer cells revealed multiple differentially methylated regions. More than 25% of islands showed complex methylation patterns either with partial methylation states defining the entire CGI or with contrasting methylation states appearing in specific regional blocks within the island. We observed that transitions in methylation state often correlate with genomic landmarks, including transcriptional start sites and intron-exon junctions. Methylation, along with specific histone marks, was enriched in exonic regions, suggesting that chromatin states can foreshadow the content of mature mRNAs. Keywords: DNA methylation profiling by massively parallel sequencing Keywords: Epigenetics Targeted examination of DNA methylation in two human cell types by combining array capture and bisulfite sequencing. In addition, this study examined two histone marks in the breast tumor cell line MDA-MB-231.
Project description:We found many binding sites for FNR under glucose fermentative anaerobic growth conditions. Also, many binding sites were identified for M-OM-^C70 under both aerobic and anaerobic growthin conditions. Descirbed in the manuscript "Genome-scale Analysis of E. coli FNR Reveals the Complexity of Bacterial Regulon Structure" Examination of occupancy of FNR adn M-OM-^C70 under aerobic and anaerobic growth in conditions.
Project description:What methylation changes are occurring during seed development largely remains unknown. To uncover the possible role of DNA methylation during the transition from seed differentiation to maturation and dormany in soybean, we characterized the methylome of whole seeds representing the differentiation (GLOB stage), maturation (early- (EM), mid- (B1) and late- (AA1) maturation stages), and dormancy (DRY stage) phases of soybean seed development using Illumina sequencing. In addition, we characterized the methylome of the mid-maturation stage embryonic axis (B1-AX) to examine methylation differences, if any, between an embryonic region compared to the whole seed. Illumina sequencing of bisulfite-converted genomic DNA from globular stage (GLOB), early-maturation stage (EM), mid-maturation stage (B1), and late-maturation stage (AA1) seeds, dormancy stage (DRY) and mid-maturation embryonic axis (B1-AX).
Project description:Global patterns of DNA methylation, mediated by the DNA methyltransferases (DNMTs), are disrupted in all cancers by mechanisms that remain largely unknown, hampering their development as therapeutic targets. Combinatorial acute depltion of all DNMTs in a pluripotent human tumor cell line, followed by epigenome and transcriptome analysis, revealed DNMT functions in unprecedented detail. DNMT3B occupancy regulates methylation during differentiation, while an unexpected interplay was discovered in which DNMT1 and DNMT3B antithetically regulate methylation and hydroxymethylation in gene bodies, a finding confirmed in other cell types. DNMT3B mediated nonCpG methylation, while DNMT3L influenced the activity of DNMT3B toward nonCpG versus CpG site methylation. Taken together, these data reveal new functional targets of each DNMT suggesting that isoform selective inhibition would be therapeutically advantageous. NCCIT cells were transfected with siRNAs against DNMT3B and a no-target control. Genomic DNA was extracted, and subsequently applied to affinity MBD-bound magnetic beads (Ribomed) to enrich methylated DNA sequences.
Project description:What methylation changes are occurring in different parts of early maturation stage seed largely remains unknown. To uncover the possible role of DNA methylation in different parts of early maturation stage seed, we characterized the methylome of seed coats,cotyledons, and the embryonic seed axis using Illumina sequencing. seed coats, cotyledon, and axis
Project description:We found many binding sites for ArcA under glucose fermentative anaerobic growth conditions. Descirbed in the manuscript "The response regulator ArcA uses a diverse binding site architechture to globally regulate carbon oxidation in E. coli" Examination of occupancy of ArcA under anaerobic growth conditions.
Project description:Inactivating mutations in the MEN1 gene predisposing to the multiple endocrine neoplasia type 1 (MEN1) syndrome can also cause sporadic pancreatic endocrine tumors. MEN1 encodes menin, a subunit of MLL1/MLL2-containing histone methyltransferase complexes that trimethylate histone H3 at lysine 4 (H3K4me3). The importance of menin-dependent H3K4me3 in normal and transformed pancreatic endocrine cells is unclear. To study the role of menin-dependent H3K4me3, we performed in vitro differentiation of wild-type as well as menin-null mouse embryonic stem cells (mESCs) into pancreatic islet-like endocrine cells (PILECs). Gene expression analysis and genome-wide H3K4me3 ChIP-Seq profiling in wild-type and menin-null mESCs and PILECs revealed menin-dependent H3K4me3 at the imprinted Dlk1-Meg3 locus in mESCs, and all four Hox loci in differentiated PILECs. Specific and significant loss of H3K4me3 and gene expression was observed for genes within the imprinted Dlk1-Meg3 locus in menin-null mESCs and the Hox loci in menin-null PILECs. Given that the reduced expression of genes within the DLK1-MEG3 locus and the HOX loci is associated with MEN1-like sporadic tumors, our data suggests a possible role for menin-dependent H3K4me3 at these genes in the initiation and progression of sporadic pancreatic endocrine tumors. Furthermore, our investigation also demonstrates that menin-null mESCs can be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null mESC-derived specialized cell types for genome-wide high-throughput studies. Genome-wide mapping of H3K4me3 and microarray gene expression profiling in TC-1 wild-type (WT) mESCs, menin-null (Men1-ko) mESCs (3.2N), pancreatic islet-like endocrine cells (PILECs) derived from WT mESCs, and PILECs derived from Men1-ko mESCs.