Single-base resolution DNA methylomes of rice and functional roles of DNA methylation
ABSTRACT: To examine the rice genome methylation landscape and assess its functional significance, we generated the first single-base resolution genome methylation maps for Oryza sativa ssp. japonica, indica and their wild relatives, Oryza rufipogon and Oryza nivara. The methylation level of rice genomes is four times higher than that of Arabidopsis. Methylation in the promoter and gene body regions have similar patterns and effects on gene expression as those in Arabidopsis but different from a previous study on rice chromosomes 4 and 10. Most interestingly, we discovered for the first time that methylation in gene transcriptional termination regions can significantly repress gene expression, and the effect is even stronger than promoter methylation, which opens a new direction in the study of epigenetic regulation of gene expressions. Through integrated analysis of genetic, methylome and expression variation between cultivated and wild rice, we found that the genetic factor reflected by DNA variations may be the major determinant for methylation patterns at the whole-genome level and that methylation variation can only account for limited expression variation of genes between cultivated and wild rice. A single young panicle from each of the cultivated rice subspecies and the two wild rice species was ground in liquid nitrogen to fine powder using mortar and pestle. Total RNAs were isolated using the RNeasy Plant Mini Kit (Qiagen). DGE-tag libraries were constructed using the DGE-Tag Profiling NlaIII Sample Prep Kit (Illumina) according to the manufacturer's instructions. This submission represents the gene expression component of the study.
Project description:To examine the rice genome methylation landscape and assess its functional significance, we generated the first single-base resolution genome methylation maps for Oryza sativa ssp. japonica, indica and their wild relatives, Oryza rufipogon and Oryza nivara. The methylation level of rice genomes is four times higher than that of Arabidopsis. Methylation in the promoter and gene body regions have similar patterns and effects on gene expression as those in Arabidopsis but different from a previous study on rice chromosomes 4 and 10. Most interestingly, we discovered for the first time that methylation in gene transcriptional termination regions can significantly repress gene expression, and the effect is even stronger than promoter methylation, which opens a new direction in the study of epigenetic regulation of gene expressions. Through integrated analysis of genetic, methylome and expression variation between cultivated and wild rice, we found that the genetic factor reflected by DNA variations may be the major determinant for methylation patterns at the whole-genome level and that methylation variation can only account for limited expression variation of genes between cultivated and wild rice. Overall design: A single young panicle from each of the cultivated rice subspecies and the two wild rice species was ground in liquid nitrogen to fine powder using mortar and pestle. Total RNAs were isolated using the RNeasy Plant Mini Kit (Qiagen). DGE-tag libraries were constructed using the DGE-Tag Profiling NlaIII Sample Prep Kit (Illumina) according to the manufacturer's instructions. This submission represents the gene expression component of the study.
Project description:Volvariella volvacea is one of a few commercial cultivated mushrooms mainly using straw as carbon source. In this study, the genome of V. volcacea was sequenced and assembled. A total of 286 genes encoding carbohydrate-active enzymes (CAZymes) in V. volvacea were identified and annotated. Among 15 fungi with sequenced genomes, V. volvacea ranks the seventh in the number of genes encoding CAZymes. In addition, the composition of glycoside hydrolases in V. volcacea is dramatically different from other basidiomycetes: it is particularly rich in members of the glycoside hydrolase families GH10 (hemicellulose degradation) and GH43 (hemicellulose and pectin degradation), and the lyase families PL1, PL3 and PL4 (pectin degradation) but lacks families GH5b, GH11, GH26, GH62, GH93, GH115, GH105, GH9, GH53, GH32, GH74 and CE12. Analysis of genome-wide gene expression profiles of 3 strains using 3'-tag digital gene expression (DGE) reveals that 246 CAZyme genes were expressed even in potato destrose broth medium. Our data also showed that the formation of a heterokaryon strain could dramatically increase the expression of a number of genes which were poorly expressed in its parental homokaryon strains. Using the 3'-tag digital gene expression (DGE), we compared the gene expression profiles among 2 homokaryotic V. volvacea strains PYd15 and PYd21, and one heterokaryotic strain H1521, which is a hybrid strain of PYd15 and PYd21.
Project description:Single-base resolution DNA methylomes have been accomplished for both Arabidopsis and human cells which have high genome methylation levels by Illumina ultra-high-throughput bisulfite sequencing technology (MethylC-Seq). Here by combining MethylC-Seq and biological replicate strategies we generated single-base resolution methylome for the silkworm which has low genome methylation levels like other insects. Our conservative estimation showed that methylcytosines (mCs) accout for about 0.11% of genomic cytosines, exclusively in CG context. The CG methylation is significantly enriched in gene bodies and positively correlated with gene expression levels, suggesting its positive role in gene transcription in silkworms. However, the well-documented functions of methylation on promoters and rDNAs in plants and mammals do not seem to have effects in insects. Methylated genes are enriched in functions involved in cellular metabolism and biosynthesis. Small RNA (smRNA) loci are also significantly enriched in gene bodies, and moreover, the smRNA loci and the predicted target sites of microRNA have high level of CG methylation, indicating functional involvement of smRNAs in the genic methylation This first methylome for silkworms provides a foundation for further studies on the epigenetic gene regulation of silkworms’ or even insects’ gene methylation. Each silk gland of 5th instar larvae of two individuals (called Biological Replicate 1 and 2, respectively) of the silkworm (Bombyx mori) strain Dazao was ground into powder in liquid nitrogen. Half of the powder from each silk gland was used to extract total DNAs using DNeasy Blood & Tissue Kit (Qiagen) and another half was used to extract total RNAs using RNeasy Mini Kit (Qiagen). We sequenced bisulfite-treated total DNA extracted from the silk glands of the two individuals, using Illumina Ultra-High-Throughput Sequencing, generating the Single-Base Resolution Methylomes. To reveal functional consequences of gene body methylation, we generated expression profiles for the two individuals’ silk glands using Digital Gene Expression tag profiling (DGE) technology, which combines classic SAGE (Serial Analysis of Gene Expression) and Illumina ultra-high-throughput sequencing technology.
Project description:Here we performed a transcriptomic study on the effect of sodium nitroprusside (SNP) on cucumber leaves under alkali stress using Solexa/Illumina's high-throughput digital gene expression (DGE) system. Two DGE libraries (from one NaHCO3-treated sample and one NaHCO3 + SNP-treated sample) were constructed, and the gene expression variations between the two samples were compared. Hundreds of differentially expressed genes were obtained by the comparison, and GO analysis of these genes suggested that many biological processes, molecular function, cellular components were related to SNP’s mitigated effect on alkaline stress. The authenticity of the DGE data was further confirmed by analyzing real-time RT-PCR using several random-selected genes. The experiment had 2 treatments: 30 mM NaHCO3 (alkali stress treatment, indicated as Na) and 30 mM NaHCO3+100 μM sodium nitroprusside (indicated as SNP). Samples from Na- and SNP-treated leaves, used for RNA isolation, were harvested, and the two corresponding tag libraries of samples were constructed in parallel. Illumina sequencing of transcripts from Na- and SNP-treated samples to get gene information for cucumber leaves in different treatments.
Project description:This research reports genome-wide measurements of genetic and epigenetic patterns of inheritance through an integrative analysis of BS-seq, RNA-seq, and siRNA-seq data in two inbred parents of the Nipponbare (NPB) and Indica (93-11) variety of rice and their hybrid offspring. We generated integrative maps of whole genome cytosine methylation profiles (BS-Seq), transcriptional profiles (RNA-seq), and small RNA profiles (sRNA-seq) to characterize two rice subspecies, Oryza sativa spp japonica (Nipponbare) and Oryza sativa spp indica (93-11) and their two reciprocal hybrid offspring using Illumina's sequencing-by-synthesis (SBS) platform .
Project description:Here we performed a transcriptomic study on complete symptom development process of CMV-infected Nicotiana tabacum using Solexa/Illumina's high-throughput digital gene expression (DGE) system. 12 DGE libraries (from six virus-infected samples and six corresponding mock-inoculated samples) were constructed, and the gene expression variations between the virus-infected sample and the mock-inoculated sample in each symptom stage were compared. Thousands of differentially expressed genes were obtained by the comparison, and KEGG pathway analysis of these genes suggested that many biological processes (such as phtosynthesis, pigment metabolism and plant-pathogen interaction) were related to the symptom development. The authenticity of the DGE data was further confirmed by analyzing real-time RT-PCR using several random-selected genes. We sequenced a cDNA library constructed from mixture of total RNA from six virus-infected samples and six mock-inoculated samples to get gene information for tobacco leaves in different symptom stages, including vein clearing, mosaic, severe chlorosis, partial recovery, total recovery and re-mosaic, and 95,916 Unigenes were obtained Tobacco leaves were inoculated by CMV-infected leaf homogenate and healthy leaf homogenate, respectivley. Six time points with different symptom stage were selected, and one virus-infect sample and one mock-inoculated sample were collected at each time. In order to average out variation of different plants, five leaves from five different plants were mixed to prepare every RNA sample. Twelve individual tag libraries of samples (six infected samples and six mock-inoculated samples) were constructed in parallel. For the gene expression analysis, the twelve samples were grouped into six groups, and each group contained a virus-infected sample and a mock-inoculated sample collected at the same time. In each group, the DGE data of virus-infected sample were compared to that of mock-inoculated sample to obtain the gene expression variations. Illumina sequencing of transcripts from virus-infected and mock-inoculated samples to get gene information for tobacco leaves in different symptom stages. In order to get more gene information, the systemically infected leaves and mock-inoculated leaves were harvested at six time points and five leaves from five different plants were collected at each time point. The RNA-Seq analysis provided gene information for mock-inoculated and virus-infected tobacco leaves in different symptom stages.
Project description:Ecological speciation is a common mechanism by which new species arise. Despite great efforts, the role of gene expression in ecological divergence and speciation is poorly understood. Here, we conducted a genome-wide gene expression investigation of two Oryza species that are evolutionarily young and distinct in ecology and morphology. Using digital gene expression (DGE) technology and the paired-end RNA sequencing (RNA-Seq) method, we obtained 21,415 expressed genes across three reproduction-related tissues at two critical developmental stages. Of them, ~8% (1717) differed significantly in expression levels between the two species and these differentially expressed genes are randomly distributed across the genome. Moreover, 62% (1064) of the differentially expressed genes exhibited a signature of directional selection in at least one species. Importantly, the genes with differential expression between species evolved more rapidly at the 5’flanking sequences than the genes without differential expression relative to coding sequences, suggesting that cis-regulatory changes are likely adaptive and play an important role in the ecological divergence of the two species. Finally, we showed evidence of significant differentiation between species in phenotype traits and observed that genes with differential expression were overrepresented with functional terms involving phenotypic and ecological differentiation between the two species, including reproduction- and stress-related characteristics. Our findings demonstrate that ecological speciation is associated with widespread and adaptive alterations in genome-wide gene expression and highlight the dominant role of regulatory evolution in ecological divergence and adaptation. We selected accessions representing typical Oryza rufipogon and O. nivara, which were sampled exclusively from South and Southeast Asia where the two species overlap. We chose to collect three types of tissues, i.e., flag leaves at the heading stage (2–7 cm above the primary branch) (L), panicles at the heading stage (H) and panicles at the flowering stage (10–15 cm above the primary branch) (F). Sample collection was repeated twice in two consecutive years (2009 and 2010) under the same controlled conditions. A total of 36 samples were sequenced by Illumina’s digital gene expression (DGE) system, with each type of tissues collected from six individuals of each species as biological replicates. To access the quality of DGE technology, we also selected six samples representing three tissues from each of two individuals (one individual per species) for paired-end RNA-Seq sequencing.
Project description:Purpose: Development of resistance to tamoxifen is an important clinical issue in the treatment of patients with breast cancer. Tamoxifen resistance may be the result of the acquisition of epigenetic regulation such as DNA methylation within breast cancer cells resulting in changed mRNA expression of genes being pivotal for estrogen dependent growth. Alternatively, tamoxifen resistance may be due to selection of preexisting resistant cells, which may exhibit cancer stem-like characteristics or a combination of the two mechanisms. Methods: To evaluate the contribution of these possible mechanisms to tamoxifen resistance, we applied modified DNA methylation-specific digital karyotyping (MMSDK) and digital gene expression (DGE) in combination with massively parallel sequencing to analyze a well-established tamoxifen resistant cell line model: MCF-7/S0.5 (tamoxifen sensitive parental cell line) and 4 high-dosage tamoxifen selected resistant offspring sublines (MCF-7/TAMR-1, MCF-7/TAMR-4, MCF-7/TAMR-7 and MCF-7/TAMR-8). MMSDK uses BssHII as mapping enzyme (DNA methylation sensitive enzyme). Both MMSDK and DGE use NlaIII and MmeI to produce 20-21 bp tag. The indexed single-end sequencing was performed by Illumina HiSeq 2000 in BGI-Shenzhen. A dynamic programming algorithm-FASTX-Toolkit implemented in Perl was used to trim the adaptor sequence. The trimmed tags were subjected to quality filtering, so that only tags with sequencing quality higher than 30 for more than 80% of the nucleotides were used for subsequent analysis. For MMSDK tag mapping, we generated a simulated reference library, i.e., BssHII reference library, by in silico enzyme digestion of the human genome (hg19, UCSC) regardless of the methylation state. This library was used as reference for subsequent mapping of the tags in the MMSDK analysis. In the DGE analysis, refMrna (hg19, UCSC) was used as reference for mapping cDNA tags. Subsequently, the Burrows–Wheeler Aligner (BWA) procedure for aligning the MMSDK and DGE tags to the simulated BssHII reference library and refMrna reference library, respectively, was applied. Results: MMSDK libraries using BssHII/NlaIII were generated from the parental tamoxifen sensitive subline MCF-7/S0.5 and the 4 TAMR cell lines: TAMR-1, TAMR-4, TAMR-7 and TAMR-8. The 5 indexed MMSDK libraries were sequenced in one lane and 1.38 Gb clean tag data for all 5 cell lines were obtained, with an average sequencing amount of ~270 Mb per library. On average, 59.5 % of the tags with mapping quality ≥ 20 were mapped back to the simulated BssHII/NlaIII reference library. DGE libraries were also generated from MCF-7/S0.5 and the 4 TAMR cell lines. The 5 indexed DGE libraries were sequenced in one lane and obtained 1.71 Gb clean tag data for all 5 cell lines with an average sequencing amount of ~340 Mb per library. On average, 40.8 % with mapping quality ≥ 20 were mapped back to the simulated NlaIII human transcriptome (refMrna reference library). Our present study demonstrates large differences in global gene expression and DNA methylation profiles between parental tamoxifen-sensitive cell line and 4 high-dosage tamoxifen treatment selected resistant sublines. The tamoxifen resistant cell lines exhibited globally higher methylation level than the parental cell line and an inverse relationship between gene expression and DNA methylation in the promoter regions were noticed. High expression of SOX2 and alterations of other SOX gene family members, E2F gene family members and RB-related pocket protein genes as well as highlighted stem cell pathways imply that cancer initiating cells/stem cells are involved in the resistance to tamoxifen. DNA methylation and mRNA expression profiles from tamoxifen sensitive parental cell line MCF-7/S0.5 and 4 high dosage of tamoxifen selected resistant offspring sublines (MCF-7/TAMR-1, MCF-7/TAMR-4, MCF-7/TAMR-7 and MCF-7/TAMR-8) were analyzed by MMSDK and DGE methods, respectively, in combination of massively parallel sequencing, using Illumina HiSeq 2000
Project description:More than 3.5 million raw DGE tags were obtained in each library. The clean tags in each sample ranged from 3.35 to 3.63 million, and the distinct clean tags ranged from 93,593 to 139,389. The 21 bp DGE clean tags were mapped to sweet potato transcripts. Then, we compared 7 libraries pair-wisely so that 21 pairs of comparisons were implemented. Among these comparisons, we found that 4,721 to 12,151 transcripts had significant changes in expression, and the average number was 9,657. We also observed a large number of specifically expressed transcripts between each two libraries. The expression profiles of those genes involved in root development and carbohydrates accumulation were characterized. Moreover, other genes of interest, such as potentially abiotic stress tolerance and insect resistance, were also characterized. 7 samples are examined: young leaves, mature leaves, stems, fibrous roots, initial tuberous roots, expanding tuberous roots and harvest tuberous roots.
Project description:The genome of V. volvacea revealed 47 genes encoding homeobox transcription factors. Of them, 8 differentially expressed homeobox transcription factors were obtained by comparing the gene expression during primordia and elongation formation. Furthermore, quantitative real-time PCR was performed on selected genes with different expression levels to demonstrate the utility of digital gene expression for gene expression profiles during fruiting body formation. The results showed that five homeobox transcription factors were up-regulated during primordia formation, and three homeobox transcription factors were down-regulated from the egg stage to elongation stage. Overall, a critical role for homeobox transcription factor was believed to contribute to the fruiting body formation. Using the 3'-tag digital gene expression (DGE), we compared the gene expression profiles in fruiting body development of V. volvacea.