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: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:Dongxiang wild rice (Oryza rufipogon Griff.) is the progenitor of cultivated rice (Oryza sativa L.) and is well known for its superior level of tolerance against cold, drought and diseases. To date, however, little is known about the salt-tolerant character of Dongxiang wild rice. To elucidate the molecular genetic mechanisms of salt-stress tolerance in Dongxiang wild rice, the Illumina HiSeq 2000 platform was used to analyze the transcriptome profiles of the leaves and roots at the seedling stage under salt stress compared with those under normal conditions. The analysis results for the sequencing data showed that 6,867 transcripts were differentially expressed in the leaves (2,216 up-regulated and 4,651 down-regulated) and 4,988 transcripts in the roots (3,105 up-regulated and 1,883 down-regulated). Among these differentially expressed genes, the detection of many transcription factor genes demonstrated that multiple regulatory pathways were involved in salt stress tolerance. In addition, the differentially expressed genes were compared with the previous RNA-Seq analysis of salt-stress responses in cultivated rice Nipponbare, indicating the possible specific molecular mechanisms of salt-stress responses for Dongxiang wild rice. A large number of the salt-inducible genes identified in this study were co-localized onto fine-mapped salt-tolerance-related quantitative trait loci, providing candidates for gene cloning and elucidation of molecular mechanisms responsible for salt-stress tolerance in rice. Leaf and root mRNA profiles of Dongxiang wild rice at the seedling stage with or without salt stress were generated by deep sequencing, on Illumina Hiseq 2000 platform.
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:Male sterility is an important trait in hybrid crop breeding. Thermo-sensitive genic male sterility (TGMS) lines, which are male-sterile at restrictive (high) temperatures but convert to male-fertile at permissive (low) temperatures, have been widely utilized in two-line hybrid rice breeding. However, the molecular mechanism underlying TGMS remains unclear. Here we show that the rice (Oryza sativa L.) thermo-sensitive genic male sterile gene 5 (tms5) locus, which in 2010 was present in cultivars occupying more than 80% (2.6 million hectares) of two-line hybrid rice-growing land in China, confers the TGMS trait through a loss-of-function mutation of RNase ZS1, resulting in failure to mediate mRNA decay of three temperature-responsive ubiquitin fusion ribosomal protein L40 genes (UbL40) genes. RNase ZS1, a member of the evolutionarily conserved endonuclease, processed tRNAs in vitro, but does not do so in vivo due to its localization in the cytoplasm. Defective RNase ZS1 in tms5 plants leads to over-accumulation of UbL401, UbL402 and UbL404 mRNAs at restrictive but not permissive temperatures. Over-expression of UbL401 and UbL404 in wild-type plants caused male sterility, whereas knockdown of UbL401 and UbL404 in tms5 plants partially restored the male fertility at restrictive temperatures. Our results uncover a novel mechanism of RNase ZS1-mediated UbL40 mRNA decay which controls TGMS in rice and has potential applications not only of rice but also of other crops. To address whether RNase ZS1 involves in mRNA metabolism, degradome sequencing was performed using RNA from young panicles of wild type (ZH11) and tms5 (Os02g12290iL1) plants grown at restrictive temperatures.
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:Hepatitis B virus (HBV) is a hepatotropic virus that can regulate many host cellular gene expressions participating in the HBV life cycle, liver inflammation and hepatocellular injury. However, the underlying mechanism of differential gene expression is not understood. We report here a genome-wide analysis of histone methylation on two histone H3 lysine residues (H3K4me3 and H3K27me3) and gene expression profiles in HepG2 and HepG2.2.15 cells. We found that specific correlation exists between gene expression and the amounts of H3K4me3 (positive correlation) and H3K27me3 (negative correlation) across the gene body. These correlations displayed three distinct modes (repressive, active and poised), reflecting different functions of these genes in the HBV life cycle, liver inflammation and hepatocellular injury. Furthermore, a permissive chromatin state of each gene was established by a combination of different histone modifications. Our findings reveal a complex regulation by histone methylation in differential gene expression and suggest that histone methylation may be responsible for the HBV life cycle, liver inflammation and hepatocellular injury induced by HBV. A large-scale analysis of gene expression of 2 different cell types (HepG2, HepG2.2.15) using a digital gene expression (DGE) tag profiling approach.
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 PaWB phytoplasma-infected Paulownia sp. using Solexa/Illumina’s high-throughput digital gene expression (DGE) system. 4 DGE libraries (from 2 virus-infected samples and 2 healthy samples) were constructed, and the gene expression variations between the PaWB phytoplasma-infected (diseased) sample and the corresponding healthy sample were compared. Thousands of differentially expressed genes were obtained by the comparison, and KEGG pathway analysis of these genes suggested that many biological processes were responded to PaWB infection. To investigate the response of Paulownia sp. to PaWB infection, we collected four samples in two groups, namely the tissue cultured group (containing healthy sample TH and diseased sample TD) and field-grown group (containing healthy sample FH and diseased sample FD). Four individual tag libraries from these samples were constructed in parallel. For the gene expression analysis, the digital gene expression (DGE) data of diseased sample were compared to that of healthy sample in each group to obtain the gene expression variations.