Unknown,Transcriptomics,Genomics,Proteomics

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OsMYB4-Regulated Transcriptional Network of Rice

ABSTRACT: The OsMyb4 transcription factor of rice represents a more recent example of a regulatory gene used in various attempts to develop stress-tolerant crops by regulon engineering. Although OsMyb4 confers tolerance to low temperature and drought by affecting the biosynthesis of compatible osmolytes and the phenylpropanoid metabolic process, the exact composition and scope of the OsMyb4 network has not been established from the previous analysis of heterologous overexpression in Arabidopsis. Further characterization of the OsMyb4 regulon at the global scale will facilitate understanding of the intricate underpinnings of a pathway independent of the DREB/CBF network. To dissect the OsMyb4 network of rice in relation to chilling stress response mechanisms, we conducted a gene expression profiling using transgenic rice overexpressing the OsMyb4 cDNA. This experiment describes the analysis of the gene expression changes as a result of supra-optimal expression of transcription factor (OsMYB4) overexpressed as transgenic in the cv. Nipponbare. Control (WT) and transgenic (OsMYB4-OX) samples were labeled with Cy3 and Cy5, respectively. No dye-swap replicates was performed in this experiment. The microarray platform used in this study was the 45k oligonucleotide microarray (www.ricearray.org), which comes in pairs of slides A and B (each containing about 50% of the total number of features). The data described in this series includes all hybridizations with slide A and with slide B. Experiments were performed with two independent biological replicates (R1 and R2) for each transgenic line.

ORGANISM(S): Oryza sativa Japonica Group

SUBMITTER: Benildo de los Reyes

PROVIDER: E-GEOD-21009 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Regulation of gene expression by reactive oxygen species (ROS) is an important component of abiotic stress signal transduction mechanisms. Previous genome-wide analysis of chilling-induced changes in gene expression suggested a potential role of H2O2 and other ROS as key signals in the induction of early response genes in japonica rice (cv. Nipponbare). Candidate regulators including bZIP-, Myb- or WRKY-types of transcription factors that were responsive to both chilling (10oC) and exogenous H2O2, and their probable target clusters (as1/ocs/TGA1-like, MybR-like element containing genes) were inferred by integrative analysis of qPCR expression profiles and ab initio promoter motif data. In an effort to define the composition and hierarchical organization of the ROS-mediated chilling response regulatory networks, we examined the effect of exogenous H2O2 (6 hours in 4mM H2O2 at 28oC and then 6 hours of recovery in water at 28oC) on the transcriptome of Nipponbare in a genome-wide scale and compared them with the chilling stress transcriptome. Results of this study were consistent with our earlier model that a ROSbZIP-as1/ocs/TGA1 regulatory module is a direct consequence of chilling-induced elevation of intracellular ROS during the initial 24 hours. Keywords: time course (response to exogenous H2O2) This experiment describes the analysis of the temporal changes in gene expression in response to exogenous H2O2 in Nipponbare rice. A total of 5 treatment regimes were examined (1, 3, and 6 hours at 4 mM H2O2 and then 3 and 6 hours of recovery in water after 6 hours of exposure to H2O2). The microarray platform used in this study was the 45K oligonucleotide microarray (www.ricearray.org) which comes in pairs of slides A and B (each containing about 50% of the total number of features). The data described in this series include all hybridizations with slide A and with slide B. All experiments were performed with two independent biological replicates (R1 and R2) for each time point.

Project description:The central role of transcriptional regulation in integrating various low temperature response mechanisms has been established in Arabidopsis, where the CBF/DREB regulon plays a prominent role during acclimation. Rice is sensitive to chilling but many japonica cultivars can survive continuous exposure to as low as 10oC for up to 7 days during the most critical stage of seedling establishment better than most indica cultivars. The transcriptional regulatory networks that define this variation have not been studied in detail as cold acclimation has been scrutinized in Arabidopsis. Towards the comparison of the compositional complexity of low temperature response regulons of rice and Arabidopsis, we used the cultivar Nipponbare for genome-wide survey of regulatory clusters by integrative analysis of promoter architectures and temporal expression profiles during exposure to 10oC at narrow time intervals. Temporal profiles revealed major clusters of genes that were induced within the initial 24 hours. These clusters were further defined by common features of having either CRT/DRE-like elements, as1/ocs-like elements or both in their promoters. Genes containing as1/ocs-like elements with or without CRT/DRE, but not those containing only CRT/DRE-like elements were induced by exogenous H2O2 but not by ABA at ambient temperature, suggesting that they belong to a potential regulon (ROS-bZIP – as1/ocs module) that responds to elevated levels of reactive oxygen species (ROS) during the initial stages of stress. Parallel analysis of transcription factors during the initial 12 hours revealed candidate regulator(s) of this putative early response regulon. Cultivar-specific expression signatures of selected members of this regulatory cluster were also positively correlated with genotypic variation in chilling tolerance. We hypothesized that the ROS-bZIP – as1/ocs cluster has important roles in configuring the transcriptome of rice seedlings during the early stages of chilling stress and it appears to be independent of ABA and functions in parallel to the CBF/DREB regulon. Keywords: time course (response to low temperature) This experiment describes the analysis of the temporal changes in gene expression in response to chilling temperature in Nipponbare rice. A total of 10 stress time points, i.e., 0.5, 2, 4, 6, 12, 18, 24, 36, 48, and 96 hrs after exposure to 10C in a growth chamber and one recovery time point, i.e., 96 hrs at 10C and then 24 hrs (24R) at ambient or control temperature (29C), were profiled with reference to a corresponding control, which was exposed to ambient temperature (29C) for the same length of time under cold stress (each stress time point has a corresponding control sample). Control and cold stress samples were labeled with Cy5 and Cy3, respectively. No dye swap replicate was performed in this experiment. The microarray platform used in this study was the 45K oligonucleotide microarray (www.ricearray.org) which comes in pairs of slides A and B (each containing about 50% of the total number of features). The data described in this series include all hybridizations with slide A and with slide B. All experiments were performed with two independent biological replicates (R1 and R2) for each time point.

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Project description:The existence of two separate lineages of Escherichia coli O157:H7 has previously been reported, and research indicates that one of these lineages (lineage I) might be more pathogenic towards human hosts. We have previously shown that the more pathogenic lineage expresses higher levels of Shiga toxin 2 (Stx2) than the non-pathogenic lineage II. To evaluate why lineage 2 isolates do not express appreciable levels of toxin, two lineage 2 isolates (FRIK966 and FRIK2000) were chosen as representatives of lineage 2 and whole genome microarrays were performed using Agilent microarrays using the E. coli O157:H7 EDL933 lineage I clinical type isolate as a reference. Microarray results were utilized to evaluate what genes and pathways might be missing or differentially expressed. Quantitative RT-PCR was utilized to validate the microarray data. Based upon the transcriptome of Escherichia coli O157:H7 EDL933 an oligonucleotide microarray, made up of 60 mers was designed. A total of 4873 genes in an 8 x 15K Agilent microarray design. Designed using a custom script, specifications for gene specific oligos were based upon various design characteristics such as temperature of melting, 3’ location, specificity, lack of repeat nucleotides, etc. (Charbonnier et al., 2005). Arrays were manufactured using Agilent Sure-print technology. Each array consisted of duplicate elements for each gene randomly distributed with Agilent control elements included. All procedures were performed according to respective manufacturer protocols. Lineage I and lineage II strains were grown overnight as described, a total of 10e7 cells were washed twice in fresh media, normalized based upon optical density, inoculated into fresh media, incubated at 37oC shaking 120 x g for 3 hours and then suspended in RNAprotect bacteria reagent (Qiagen Inc., Valencia, CA.). Total RNA was extracted using RNeasy Bacteria Mini Kit (Qiagen Inc., Valencia, CA.) and trace amounts of DNA were removed using RNase-Free DNase Set (Qiagen Inc., Valencia, CA.). RNA was quantified using Nanodrop system (NanoDrop Technologies, Wilmington, DE) and quality confirmed by electrophoresis on a Bio-rad Experion system (BioRad XXX). For each sample, 10 ug of RNA were labeled with either CyDye3-dCTP or CyDye5-dCTP (Perkin Elmer) using the LabelStar kit (Qiagen Inc., Valencia, CA.) and Random nonamers (Integrated DNA Technologies ). Labeled cDNA were hybridized to the microarray using Agilent Hi-RPM hybridization solution in an Agilent Hybridization chamber (Agilent.). A total of 8 arrays, each with duplicate elements for each gene, alternating dye swap for each replicate (4 biological replicates), were analyzed to obtain genes that were consistently and differentially regulated in comparison to EDL933, while limiting false discover rate (FDR) below a stringent 5% (Benjamini and Hochberg, 1995).

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Project description:We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis. Genomic array design Microarrays were designed as described below and manufactured by Agilent Technologies (www.agilent.com). Further information on design can be found at http://jura.wi.mit.edu/bioc/gbell/zfish_chip/. Selection of transcription start sites and identification of promoter sequences We interrogated 5 databases: Ensembl, VEGA, Refseq, ZGC full length clones and a database provided by Dr. Leonard Zon (Harvard Medical School, Boston, USA) in order to assemble an extensive list of zebrafish transcripts. The Zon lab database is a hand-curated database of zebrafish genes that have homologues in other species. We included all transcripts that appeared in the manually annotated databases (VEGA, Zon) and in the ZGC full length database. We also identified genes present in any 2 of the 5 databases and included those not already selected. The transcripts were mapped to the zebrafish genome (Zv4, June 2004) obtained from UCSC Bioinformatics (http://genome.ucsc.edu) and the transcription start site (TSS) for each transcript was determined. Transcripts with TSSs within 500bp were clustered into a transcriptional unit (TU) and promoter regions were identified relative to the most upstream TSS. This resulted in the identification of 13,413 TUs and corresponding promoter regions. Each promoter region was extracted and masked for repetitive sequence by RepeatMasker. If the promoter region contained a gap the upstream sequence was also masked. Information on the transcriptional units that were included in the final design can be found at http://jura.wi.mit.edu/bioc/gbell/zfish_chip/. Selection of oligonucleotides 60-mer oligonucleotide probes representing the region between 1.5kb upstream and 0.5kb downstream of the annotated TSS of each transcriptional unit were then designed. Although transcription factors and other DNA binding proteins are known to regulate genes from distances of greater than -1.5kb or + 0.5kb, much information can be gained from regions close to the TSS [45], and the H3K4Me3 mark studied in this paper is found at the most 5â end of a gene, close to the TSS. Selection of 60-mers for the microarrays was essentially as described in [14] using the Zv4 build of the zebrafish genome and a locally customized version of ArrayOligoSelector. 60-mers were chosen so that promoter regions contained approximately one probe every 250bp with a maximum distance between probes for each promoter region set at 600bp. In cases where only one probe could be designed for a particular TU these were not included in the final design. This process yielded 80,839 probes for 11,171 promoter regions We also incorporated several sets of control probes, both positive and negative. On each array there are 1090 probes designed against âgene desertâ regions, which are genomic regions that are unlikely to be bound by transcriptional regulators, and 270 probes designed against Arabidopsis thaliana genes, which are not present in the zebrafish genome (by BLAST). In addition, because our main motivation for making these microarrays is to identify mesodermally-regulated genes we included 7 genes expressed in mesoderm during gastrulation as positive controls (wnt11, flh, vent, msgn1, myod, fgf8, pcdh8). Probes designed against these promoters, which flank from 3-4kb around each TSS, are arrayed 2-4 times on each slide. Since these genes are expressed at gastrula stages to varying degrees, they also serve as a positive controls in this study. Finally there are 2256 controls added by Agilent and a variable number of blank spots. These probes were divided between two microarray slides each with 44,290 features. We refer to these two microarray slides as the âproximal promoter setâ. A proximal promoter set based on these designs as well as an expanded set of 9 slides which contain regions from â9kb to + 3kb relative to the TSS, are available by contacting Agilent (www.agilent.com) or by downloading the design files from http://jura.wi.mit.edu/bioc/gbell/zfish_chip/ for self-manufacture.

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OsMYB4-Regulated Transcriptional Network of Rice

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