Sub1 occupancy in yeast cells in exponential growth condition
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE11054 | GEO | 2009/08/27
SECONDARY ACCESSION(S): PRJNA107043
REPOSITORIES: GEO
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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 Chromatin immunoprecipitation Chromatin immunoprecipitations were performed essentially as described by Ren et al. (2000) with some modifications described below. Briefly, overnight yeast cultures were used to inoculate fresh YPD medium at 0.1 OD600. The cells were grown in 100 mL of YPD to 1 OD600 and fixed with formaldehyde at a final concentration of 1% for 15 min at room temperature. Fixation was stopped by the addition of glycine (final concentration 340 mM) and incubation for 5 min. The fixed cells were harvested by centrifugation and washed twice with cold TBS. The cell pellets were frozen and stored at –80°C for further use. The cell pellets were thawed, resuspended in 700 µL of lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 1 mM PMSF, O Complete protease inhibitor (Roche)) and transferred to 1.5 mL Eppendorf tubes. 0.5 mL glass beads (425-600 µm, Sigma) were then added to each tube, and the yeast cells were lysed at 4°C for 2 hours using an Eppendorf Thermomixer Comfort at 1400 rpm. Each tube was pierced at the bottom and the cell lysate was collected by centrifugation in a fresh tube. The lysate was then sonicated 4 times for 10 s with 20 s cooling on ice between each sonication. The power of the Vibra Cell™ sonicator (Sonics & Materials Inc., Danbury, Connecticut, USA) was set to 4 with 60% duty cycle. Sonication resulted in chromatin fragments of 0.25–1 kb in size with a mean size of 600 base pairs as determined by gel electrophoresis. After 5 min of centrifugation in 1.5 mL Eppendorf tubes at 15 000 rpm, the supernatant (named whole cell extract, WCE) was transferred to another tube on ice. The preparation of magnetic beads, immunoprecipitation (with anti-HA 12CA5 or anti-myc 9E10), elution from beads and reversal of crosslinking, DNA precipitation, DNA blunting and ligation of blunted DNA to linkers were done exactly as described in Ren et al. (2000). Construction of yeast microarrays The S. cerevisiae ORF and intergenic regions were amplified from S288C genomic DNA with oligonucleotides from ResGen. The primers allow the amplification of the sequence located on either side of elements such as open reading frames, tRNAs, small nuclear RNAs, Ty elements, solo δ, etc. A complete description of the primers can be obtained on the web site (ftp://ftp.resgen.com/pub/genepairs/yeast_intergenic). The PCR products were purified by ethanol precipitation and their size and concentration were measured by agarose gel electrophoresis. 95 % of the regions were correctly amplified (as evidenced by gel electrophoresis) and purified at a mean plate concentration of 70 ng/µL. 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 %. The final success rate is 93-94 %. LM-PCR, probe labeling and hybridization Ligation-mediated PCR was done as described by Ren et al. (2000) except that amino-allyl conjugated dUTP (150 µM final) was used instead of Cy3-dUTP or Cy5-dUTP and only 30 cycles of PCR were performed. After PCR amplification, 5 μl of the reaction mixtures was run on 1.5% agarose gel to measure the amount of amplified DNA. The PCR product size ranged from 200 to 1000 bp with an average size of 300 bp. The PCR products were purified using a Microcon YM-30 filter (Amicon/Millipore). Amino-allyl modified DNA was 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. 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). Data from three independent experiments were compiled. ChIP-chip spot intensities have been normalized using the weighted median as implemented in the normalizeWithinArrays function of the Limma package. Normalized measures served to compute the log2-ratio for each probe. To exhibit enriched probes, we have used 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, but we have considered a one-sided alternative hypothesis, since we expect higher intensity levels in the IP-enriched hybridizations than in the negative control hybridizations.
2009-08-27 | E-GEOD-11054 | biostudies-arrayexpress
Project description:During transcription the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region transcribed by Pol I and over the 5S rDNA transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5?-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on highly expressed protein-coding genes in the wild-type, notably over the second exon of spliced ribosomal protein genes. ChIP-seq of RNA-DNA hybrids using antibody S9.6
2014-11-03 | E-GEOD-53420 | biostudies-arrayexpress
Project description:ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of transcription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome-wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occupancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC catalytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over transcribed regions. ChIP-chip experiments to measure Sth1, Rpb3 and H3 occupancy in WT and various mutants (histone acetyltransferase and Pol II CTD kinase mutants). The histone H3 and Rpb3 occupancy were also measured in cells upon Sth1 depletion.
2014-11-24 | GSE62519 | GEO
Project description:During transcription the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region transcribed by Pol I and over the 5S rDNA transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5?-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on highly expressed protein-coding genes in the wild-type, notably over the second exon of spliced ribosomal protein genes.
2014-11-03 | GSE53420 | GEO
Project description:ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of transcription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome-wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occupancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC catalytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over transcribed regions. ChIP-chip experiments to measure Sth1, Rpb3 and H3 occupancy in WT and various mutants (histone acetyltransferase and Pol II CTD kinase mutants). The histone H3 and Rpb3 occupancy were also measured in cells upon Sth1 depletion. The WT and mutant strains were grown in Synthetic complete or YPD media to an O.D. 600 of 0.6-0.8. For inducing Gcn4, the cells grown in SC were treated with Sulfometuron methyl for 20-25 minutes and process for chromatin immunoprecipitation using antibodies again Myc, Rpb3 or histone H3.
2014-11-24 | E-GEOD-62519 | biostudies-arrayexpress
Project description:ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of transcription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome-wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occupancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC catalytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over transcribed regions. In these experiments, we have analyzed Sth1 (catalytic subunit of the RSC chromatin remodeling complex) enrichment to the transcribing genes.
2014-11-24 | GSE62516 | GEO
Project description:Nucleolar ribosomal DNA (rDNA) repeats control ribosome manufacturing. rDNA harbors a ribosomal RNA (rRNA) gene and an intergenic spacer (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding the rRNA components of ribosomes. Pol II at the IGS induces rRNA production by preventing Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli. At the IGS, Pol II regulatory processes and whether Pol I function can be beneficial remain unknown. Here, we identify IGS Pol II regulators, uncovering nucleolar optimization via IGS Pol I. Compartment-enriched proximity-dependent biotin identification (compBioID) showed enrichment of the TATA-less promoter-binding TBPL1 and transcription regulator PAF1 with IGS Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I and its ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the interactome of nucleolar Pol II and show its control by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and operation.
2024-10-09 | GSE261857 | GEO
Project description:Set2p, which mediates histone H3 Lysine 36 dimethylation (H3K36me2) in Saccharomyces cerevisiae, has been shown to associate with RNA polymerase II (RNAP II) at individual loci. Here, ChIP-chip experiments normalized to general nucleosome occupancy reveal that nucleosomes within open reading frames (ORFs) and downstream non-coding chromatin were highly dimethylated at H3K36, and that Set2p activity begins at a stereotypic distance from the initiation of transcription genome-wide. H3K36me2 is scarce in regions upstream of divergently transcribed genes, telomeres, silenced mating loci, and regions transcribed by RNA polymerase III, providing evidence that the enzymatic activity of Set2p is restricted to its association with RNAP II. The presence of H3K36me2 within ORFs correlated with the "on" or "off" state of transcription, but the degree of H3K36 dimethylation within ORFs did not correlate with transcription frequency. This provides evidence that H3K36me2 is established during the initial instances of gene transcription, with subsequent transcription having at most a maintenance role. Accordingly, newly activated genes acquire H3K36me2 in a manner that does not correlate with gene transcript levels. Finally, nucleosomes dimethylated at H3K36 appear to be refractory to loss from highly transcribed chromatin. Thus H3K36me2, which is highly conserved throughout eukaryotic evolution, provides a stable molecular mechanism for establishing chromatin context throughout the genome by distinguishing potential regulatory regions from transcribed chromatin. Keywords: ChIP-chip
2005-10-01 | GSE2991 | GEO
Project description:Following radiation damage, cells resume ribosome biogenesis for survival. We combined chromatin immunoprecipitation (ChIP) with mass spectrometry to identify key binding partners of RPA194 (the major subunit of RNA polI) at the rDNA loci in response to irradiation.
2023-07-20 | PXD041472 | Pride
Project description:ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of transcription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome-wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occupancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC catalytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over transcribed regions. In these experiments, we have analyzed Sth1 (catalytic subunit of the RSC chromatin remodeling complex) enrichment to the transcribing genes. The cells (WT and gcn4M-NM-^T) harboring STH1-MYC allele were treated by SM for 20 minutes to induce Gcn4 regulated genes. The chromatin extracts were prepared and subjected to chromatin immunoprecipitation using anti-Myc antibodies. The ChIP DNA as well the corresponding input DNA were biotinylated and hybridized to the Affymetrix tiling Arrays. Chromatin samples from two different cultures were used in this analysis.
2014-11-24 | E-GEOD-62516 | biostudies-arrayexpress