Gene expression in Delta Sub1 with and without 4NQ0
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE11124 | GEO | 2011/04/01
SECONDARY ACCESSION(S): PRJNA106905
REPOSITORIES: GEO
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Project description:Microarrays analysis was undertaken to compare mRNA levels in exponentially growing wild-type and sub1Δ cells, or after 1 h treatment with 4NQO. After spot quantification and normalization, only few genes were found to be differentially expressed when the two strains were exponentially grown in rich medium. In contrast, the distribution of gene expression ratios was clearly different upon 4NQO treatment showing that the transcriptional response to DNA damages was affected in the absence of Sub1. Most of the differentially expressed genes were up-regulated in sub1Δ cells, suggesting a global repression effect of Sub1. About half of them were bound by Sub1 demonstrating that Sub1 occupancy correlates with gene regulation. A GoTermFinder analysis revealed an enrichment in genes implicated in cell cycle process for the down-regulated genes. The up-regulated genes were enriched in ribosome biogenesis and cell wall structure GO categories, confirming that most of the genes bound by Sub1 were up-regulated in sub1Δ cells upon 4NQO treatment. A third GO category corresponded to genes encoding proteins involved in amino acid metabolism whose expression is under the control of the Gcn4 transcriptional activator. This GO category could reflect an indirect effect since the GCN4 gene is bound by Sub1, up-regulated in sub1Δ cells and induced at the translational level in response to DNA-damaging agents. Keywords: Transcriptomic Genomic Comparative Cell culture and RNA extraction. Wild-type (yPH500) or sub1Δ cells were grown to exponential phase (OD600 = 1) in YPD medium at 30°C and an aliquot of cells was collected to serve as the time-zero reference. After the addition of 4NQO (1 µg/µl), the cells were further incubated for 1h. The cells were harvested by centrifugation and resuspended in 50 mM Na acetate, pH 5.3, 10 mM EDTA. Total RNA was isolated by heating and freezing the cells in the presence of SDS and phenol as described (Schmitt et al., 1990). Preparation of labeled cDNAs Labeled cDNAs were synthesized from 20 to 30μg total RNA using an indirect labeling protocol adapted from P.Brown (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm) with the Superscript II reverse transcription kit (Gibco-BRL) and amino-allyl dUTP. lAmino-allyl modified DNA was purified on Microcon filters (Amicon) recovered with 20 µL of H2O and the DNA was lyophilized. The DNA pellet was resuspended in 9 µL of 100 mM sodium bicarbonate, pH 9.0. This sample DNA was used to dissolve a dry pellet of monofunctional NHS-ester Cy3 or Cy5 (1/16e of the quantity delivered from the mono-reactive dye pack PA23001 or PA25001 respectively from Amersham) and the mixture was incubated for 1 hour at room temperature in the dark. The coupling reaction of the Cy dyes to the amino-allyl dUTP was stopped by quenching with the addition of 4.5 µL of 4M hydroxylamine (Sigma) and the solution was further incubated for 15 min in the dark. Cy3-immunoprecipitated DNA and Cy5-WCE control DNA were mixed and unincorporated/quenched Cy dyes were removed using a Microcon YM-30 filter (Amicon/Millipore). DNA was recovered with 60 µL of TE and ethanol precipitated. Construction of ORF-microarrays S. cerevisiae open reading frames (ORF), reamplified using a pair of universal primers from 6144 full-length ORFs (1 ng) amplified from genomic DNA by Research Genetics. The PCR products were purified by ethanol precipitation, and their size and concentration were measured by agarose gel electrophoresis. 92.4% of the ORFs were correctly amplified and purified. The purified DNAs were spotted onto amino-silane coated glass slides (GAPS II, Corning) using an automated arrayer (MicroGrid II, BioRobotics) with a spotting success percentage higher than 96 %.. Prehybridization and hybridization. Prehybridization and hybridization conditions were those described on P. Brown's web site (http://brownlab.stanford.edu/protocols.html) with few minor modifications. The labeled precipitated DNA samples were recovered in hybridization buffer (50% formamide, 5X Denhardt’s, 0.5% SDS, 7X SSPE, 10 µg herring sperm DNA) and hybridized to the DNA microarray in a Corning slide chamber at 42°C overnight. Arrays were washed at room temperature once with 0.1X SSC + 0.1% SDS, twice with 0.1X SSC, then dried by centrifugation. Data analysis Hybridized arrays were scanned using a GenePix 4000A scanner (Axon Instruments, Inc.) and fluorescence ratio measurements were determined with the GenePix Pro 6.0 software (Axon Instruments, Inc.). Array analyses were undertaken using the Limma package (Smyth, 2005) from the R/Bioconductor software (R-Development-Core-Team, 2007). Microarray spot intensities have been normalized by subtracting the background and using the LOWESS method (Cleveland, 1979) with the smooth parameter set to 0.33 as recommended (Quackenbush, 2002). Data from four technical replicates corresponding to two independent experiments were compiled. Normalized measures served to compute the ratios of Cy3/Cy5 intensity and the associated log2-transform (denoted log2-ratios) for each gene. To identify genes with a log2-ratio significantly different between the mutant and wild-type strains, p-values were calculated for each gene using a moderated t-test. The moderated t test applied here was based on an empirical Bayes analysis and was equivalent to shrinkage (or expansion) of the estimated sample variances towards a pooled estimate, resulting in a more stable inference.
2011-04-01 | E-GEOD-11124 | biostudies-arrayexpress
Project description:A genome-wide location analysis by ChIP on chip of the gene occupancy by Sub1 was undertaken to define the gene targets of Sub1 in vivo. Chromatin immunoprecipitation (ChIP) assays were performed on epitope-tagged Sub1-3HA cross-linked chromatin from exponentially growing cells. Immunopurified DNA and DNA from whole-cell extracts were fluorescently labelled and competitively hybridized to DNA microarrays harbouring ORFs and intergenic regions. The ratio of fluorescence intensities at each site in the microarray provided a measure of the extent of Sub1 binding to a specific genomic locus. Data from three independent experiments were compiled. Many loci were found to be significantly enriched in active growth conditions where Sub1 is not essential for cell viability. Approximately one-fourth of the enriched loci were located within ORF and the others corresponded to intergenic regions and to genes encoding non-translated RNAs. The ACT1, PMA1, PYK1, ADH1 and snoRNA genes previously identified as DNA targets of Sub1 were indeed enriched in our data. Sub1 was also preferentially bound to a subset of Pol II-transcribed genes encoding constituents of the cell wall, the nucleosome and the ribosome. Remarkably, these Pol II genes represented only two-third of the enriched loci. The other ones corresponded to Pol III-transcribed genes present on the arrays or to the intergenic regions and ORFs adjacent to these genes, suggesting the association of Sub1 to all Pol III-transcribed genes in conditions of active growth. The arrays used had a poor coverage of the rDNA gene locus. Nevertheless, we found a significant enrichment of the different loci corresponding to that region suggesting that Sub1 associates at many locations throughout the rDNA gene. Altogether, the results suggested that Sub1 could be a regulator of all three transcription systems. Keywords: ChIP-Chip
2009-08-27 | GSE11054 | GEO
Project description:Gcn4 is a yeast transcriptional activator induced by amino acid starvation. ChIP-seq analysis revealed 546 genomic sites occupied by Gcn4 in starved cells, representing ~30% of all Gcn4 binding-motifs. Deviation from the consensus motif and nucleosome occupancy are key negative determinants of Gcn4 binding. Surprisingly, only ~40% of the bound sites are in promoter regions, and only ~50-67% of these activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining ~300 Gcn4-bound sites are within coding sequences (CDS), with ~75 representing the only bound sites near Gcn4-induced genes. Many such unconventional sites map between divergent antisense and sub-genic sense transcripts induced within CDS, adjacent to induced TBP peaks—consistent with Gcn4 activation of cryptic, bidirectional internal promoters. Mutational analysis confirms that Gcn4 sites within CDS can activate sub-genic and full-length transcripts from the same or adjacent genes, showing that functional Gcn4 binding is not confined to promoters.
2018-02-26 | GSE110413 | GEO
Project description:Gcn4 is a yeast transcriptional activator induced by amino acid starvation. ChIP-seq analysis revealed 546 genomic sites occupied by Gcn4 in starved cells, representing ~30% of all Gcn4 binding-motifs. Deviation from the consensus motif and nucleosome occupancy are key negative determinants of Gcn4 binding. Surprisingly, only ~40% of the bound sites are in promoter regions, and only ~50-67% of these activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining ~300 Gcn4-bound sites are within coding sequences (CDS), with ~75 representing the only bound sites near Gcn4-induced genes. Many such unconventional sites map between divergent antisense and sub-genic sense transcripts induced within CDS, adjacent to induced TBP peaks—consistent with Gcn4 activation of cryptic, bidirectional internal promoters. Mutational analysis confirms that Gcn4 sites within CDS can activate sub-genic and full-length transcripts from the same or adjacent genes, showing that functional Gcn4 binding is not confined to promoters.
2018-03-23 | GSE107532 | GEO
Project description:Deletion of several ribosomal proteins genes (RPKOs) has been shown to extend the lifespan of Saccharomyces cerevisiae in a Gcn4-dependent manner. To characterize the underlying mechanisms, we systematically analyzed the gene expression of both short- and long-lived RPKO strains at multiple levels. We found that up-regulation of amino acid biosynthesis and global down-regulation of protein synthesis are hallmarks of long-lived strains. We provide direct evidence that gene expression changes observed in long-lived strains result from translational up-regulation of GCN4 mRNA via skipping of upstream open reading frames (uORFs), in turn due to slow/defective ribosome assembly. We further demonstrate that Gcn4 acts as a transcriptional repressor on promoters of translation-related genes, thereby globally reducing protein synthesis. Our data suggest that the Gcn4-dependent increase in lifespan can be attributed partially to its ability to dampen the translation capacity of the cell, thereby engaging a well known mechanism of longevity.
2017-07-06 | PXD004760 | Pride
Project description:Intervention type:DRUG. Intervention1:Huaier, Dose form:GRANULES, Route of administration:ORAL, intended dose regimen:20 to 60/day by either bulk or split for 3 months to extended term if necessary. Control intervention1:None.
Primary outcome(s): For mRNA libraries, focus on mRNA studies. Data analysis includes sequencing data processing and basic sequencing data quality control, prediction of new transcripts, differential expression analysis of genes. Gene Ontology (GO) and the KEGG pathway database are used for annotation and enrichment analysis of up-regulated genes and down-regulated genes.
For small RNA libraries, data analysis includes sequencing data process and sequencing data process QC, small RNA distribution across the genome, rRNA, tRNA, alignment with snRNA and snoRNA, construction of known miRNA expression pattern, prediction New miRNA and Study of their secondary structure Based on the expression pattern of miRNA, we perform not only GO / KEGG annotation and enrichment, but also different expression analysis.. Timepoint:RNA sequencing of 240 blood samples of 80 cases and its analysis, scheduled from June 30, 2022..
| 2612481 | ecrin-mdr-crc
Project description:In order to clarify the molecular mechanisms of drought tolerance between different maize (Zea mays L.) varieties at the protein level, iTRAQ (the isobaric tags for relative and absolute quantitation) quantitative proteomics were used for the comparative analysis of protein expression in the seedling roots of the drought-tolerant Chang 7-2 and drought-sensitive TS141 maize varieties under 20% PEG 6000 (polyethylene glycol 6000)-simulated drought stress. A total of 7723 proteins were identified using iTRAQ in the maize seedling roots. We detected 39371 peptides and 30148 unique peptides. The identified protein molecular mass was mainly distributed between 10–60 kDa. COG analysis divided these proteins into 24 categories, among which general function prediction contained the largest number of proteins (19.1%), followed by posttranslational modification, protein turnover, and chaperones (10.52%). In the identified differentially expressed proteins, 1552 of which were significantly differentially expressed. Of these, 1249 were differentially expressed in Chang 7-2 following drought stress, 577 of which were up-regulated and 672 were down-regulated. Four hundred and twenty five differentially expressed proteins were identified in TS141, 249 of which were up-regulated and 176 were down-regulated. Of these proteins, 32 demonstrated opposite expression levels in the two varieties, and there were 56 up-regulated proteins that were common between the two varieties. Comparing the Chang 7-2 treatment group and control group, the DEPS in the cellular component category were mainly enriched in cytoplasmic membrane-bounded vesicles, cytoplasmic vesicles, membrane-bound vesicles and vesicles; in the molecular function category, the DEPs were mainly enriched in cation binding and metal ion binding; and in the biological process category, the major enriched categories included phosphate-containing compound metabolic process, single-organism transport cellular component organization or biogenesis, carbohydrate metabolic process, and cellular component organization. Comparing the TS141 treatment group and control group, the DEPS in the cellular component category were primarily enriched in cytoplasmic membrane-bounded vesicles, cytoplasmic vesicles, membrane-bound vesicles, and vesicle; in the molecular function category, the DEPs were mainly enriched in cation binding and metal ion binding; and in the biological process category, the major enrichment categories included oxidation-reduction process and carbohydrate metabolic process. In Chang 7-2, the differentially expressed proteins were associated with ribosome pathway, glycolysis/gluconeogenesis pathway, and amino sugar and nucleotide sugar metabolism. In TS141, the differentially expressed proteins were associated with metabolic pathway, phenylpropanoid biosynthesis pathway, and starch and sucrose metabolism.
2019-11-13 | PXD012344 | Pride
Project description:Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences now serving and promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences, and allows the characterization of TF binding determinants within and outside of core binding sites. Binding experiments were carried out with one of two input libraries; Lib1 (also referred to as “main lib”) includes designed sequence variants of length 150bp, and Lib2 (also referred to as “constant flanks lib”) includes designed sequence variants of length 200bp. The sequences of the variants of interest can be found in the second column of the processed data file (BunDLE-seq_data, see 3 excel sheets). EXP1, EXP2 and EXP3 were carried out using Lib1, and EXP4 was carried out using Lib2. Binding experiments were carried out with different proteins (in different concentrations) – DNA was extracted from bound bands that were formed on the gel and amplified with a primer with a unique 5bp upstream tail specifying the identity of the originating band (also referred to as “band barcode”). Data from the following bands was used in the analysis: EXP1_band1, no-protein band – with band barcode “AACGA”, EXP1_band2, single Gcn4 (conc 1:8) bound band* – with band barcode “TCGTA” , EXP1_band3, histones bound band – with band barcode “AGATA”, EXP2_band1, no-protein band – with band barcode “AACGA”, EXP2_band2, single Gcn4 (conc 1:8) bound band* – with band barcode “AGATA”, EXP2_band2, single Gcn4 (conc 1:7) bound band – with band barcode “CACTT”, EXP2_band2, single Gcn4 (conc 1:6) bound band – with band barcode “TCGTA”, EXP2_band2, single Gcn4 (conc 1:5) bound band – with band barcode “CATCA”, EXP2_band2, single Gcn4 (conc 1:4) bound band – with band barcode “TATTA”, EXP2_band2, single Gcn4 (conc 1:3) bound band – with band barcode “AGTCA”, EXP2_band2, single Gcn4 (conc 1:2) bound band – with band barcode “ACGAA”, EXP2_band2, single Gcn4 (conc 1:1) bound band – with band barcode “ACAGA”, EXP2_band2, two Gcn4 (conc 1:1) bound band – barcoded with “GTAGA”, EXP3_band1, no-protein band – with band barcode “GCATA”, EXP3_band2, single Gal4 (conc 1:1) bound band – with band barcode “GATGT”, EXP3_band3, single Gal4 (conc 3:1) bound band – with band barcode “GTTCA”, EXP3_band4, two Gal4 (conc 3:1) bound band – with band barcode “CTCAA”, EXP4_band1, no-protein band – with band barcode “GAGTA”, EXP4_band2, single Gcn4 (conc 1:3) bound band – “TACCA”, EXP4_band3, no-protein band – with band barcode “TCGTT”, EXP4_band3, single Gal4 (conc 3:1) bound band – with band barcode “ACATC”, *biological replicates
2015-03-04 | E-GEOD-66143 | biostudies-arrayexpress
Project description:A total of 432 genes were found to be differentially expressed in M1SF370 bacterial population internalized in Detroit 562 human pharyngeal cells when compared with the same strain incubated in the absence of Detroit 562 cells. While most of them (349/432 i.e. 80.8%) were up regulated, 83 genes were down regulated contributing to 19.2% of the total differentiated genes. The major contributor of the latter category was phage-related genes (35 genes). Almost ¼ of these genes (106) belonged to a category of Unknown or possible predicted function. Most notably, up-regulated genes belonged to amino acid transport , cell division, cell envelope biogenesis, DNA replication correlated well with up-regulated 67 genes belonged to translation and ribosomal structure. Further, up-regulation of 12/15 virulence-related genes indicated that human host cell internalized bacteria are highly virulent as compared to laboratory grown culture in test-tubes.
2011-06-10 | GSE27846 | GEO
Project description:The anabolic androgen 17β-trenbolone (TB) can cause masculinization and reduce fecundity of fish. However, the underlying mechanisms of various biological pathways including metabolism, biosynthesis etc. are largely unknown. Here, we evaluated the effects of TB using the medaka DNA microarray representing 36,398 genes. Larval medaka, Oryzias latipes, (within 24 hrs posthatch) were exposed to TB at various concentrations (2, 6, 20, 60, 100, 200 ng/L) for up to 7 days. Dose-response relationships in gene expression levels of the categorized genes were analyzed using the cumulative chisquared method. Microarray analyses of the TB-exposed larvae showed that 117 and 32 genes were determined as up and down-regulated genes, respectively. The most significant GO term for biological process identified within this gene list was lipid metabolic process, which contained 26 genes in up-regulated genes. In this category, “cholesterol biosynthetic process” was highlighted as an important subcategory with 15 genes, including hydroxymethylglutaryl-CoA synthase cytoplasmic, squalene monooxygenase, lanosterol synthase etc. RT-PCR measurements in these genes were consistent with the microarray results in the direction and magnitude of these changes in gene expression. On the other hands, in the category of “sexual differentiation and development”, genes related to hypothalamic-pituitary-gonadal (HPG) axis were not affected by TB treatment except for one gene encoded to cytochrome P450 19A1. Genes related to oogenesis, such as choriogenins and vitellogenins were weakly down regulated at 2-200 ng/L of TB. Our findings demonstrate that genes encoding cholesterol synthesis pathway via the mevalonate pathway were controlled by TB in larval medaka.
2013-02-14 | GSE35908 | GEO