Protein kinase C coordinates histone H3 phosphorylation and acetylation
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae S288c
SUBMITTER: Leo Zeef
PROVIDER: E-MTAB-3359 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Transcriptional profiling of a Saccharomyces cerevisiae protein kinase Pkc1p temperature sensitive mutant on cells synchronised in S phase and then released. The Mcm1p-Fkh2p-Ndd1p complex in Saccharomyces cerevisiae controls the cyclical expression of the CLB2 cluster of genes at the G2/M phase transition. The activity of this complex is known to be positively controlled by cyclin-CDK and polo kinases. The current experiment aimed to demonstrate that the protein kinase Pkc1p works in the opposite manner to inhibit the activity of the Mcm1p-Fkh2p-Ndd1p complex and the expression of its target genes.
2013-03-14 | E-MEXP-3667 | biostudies-arrayexpress
Project description:To identify atrophy genes directly targeted by Bcl-3 transactivator at a genome wide level, we performed whole transcript expression array and ChIP-seq for muscles from weight bearing or 5-day hind limb unloaded mice. Genes that showed increased expression with unloading and a Bcl-3 peak in the promoter (from ChIP-seq data) were considered as Bcl-3 direct targets during disuse atrophy. Using ChIP array, we identified 241 direct targets for Bcl-3. Our data describe Bcl-3 as a global regulator both of the proteolysis and the change in energy metabolism that are essential components of muscle atrophy due to disuse. Disuse skeletal muscle atrophy was induced by hind limb unloading. Weight bearing (WB) or 5-day hind limb unloaded (HU) muscles were harvested for total RNA isolation and processed for whole transcript expression profiling. We chose to examine gene expression and Bcl-3 binding from 5-day unloaded muscles because our previous time course study of disuse atrophy suggested that most genes are differentially regulated at this time point, and thus, would best represent the time for Bcl-3 binding to the gene targets of the NF-kB transcriptional network.
2012-11-10 | E-GEOD-40578 | biostudies-arrayexpress
Project description:We applied serial ultrafiltration and limited proteolysis-coupled mass spectrometry (LiP-MS) to generate an atlas of peptides that act as markers for the formation or dissolution of protein-protein interactions (PPIs). Applied to Saccharomyces cerevisiae, the approach yielded 6,441 candidate PPI markers from 1,086 proteins, including 1,072 markers of known interfaces from 272 proteins. These include peptides mapping to both validated and putative protein binding interfaces, as well as markers of structural changes that accompany PPIs. The approach is based on detecting differences in protease susceptibility between the protein complex-bound and monomeric forms. We then used these peptide markers to track changes in protein-protein interactions in LiP-MS analyses of unfractionated lysates of yeast grown under hydroxyurea (HU) stress compared to control conditions. We captured known and novel protein complex rearrangements, supporting the key role played by Spt-Ada-Gcn5 acetyltransferase (SAGA) and the activity of its acetyltransferase Gcn5 in the stress response. We validated our observed rearrangement of SAGA with an AP-MS experiment comparing Ada3 pulldowns of yeast grown under HU and control conditions. We further examined how protein complex rearrangements change in Gcn5 mutant cells; we found a set of protein complexes that depend directly or indirectly on Gcn5 activity and identified a link between Gcn5 activity and the regulation of P-bodies.
2024-10-17 | PXD055566 | Pride
Project description:The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres, where it provides the cohesive counter-force to bi-polar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometeres. This M-bM-^@M-^Xcentromere breathingM-bM-^@M-^Y presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast S. cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, that has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is re-loaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Keywords: ChIP-chip SAMPLES Origin of each biological sample: Saccharomyces cerevisiae (W303background) Growth conditions: Cells containing the MET3-CDC20 allele were grown at 25M-BM-0C in SC medium lacking methionine with 2% raffinose or 2% glucose as the carbon source (Uhlmann et al. 2000). Cultures were synchronized in G1 with M-NM-1-factor (5 M-NM-<g/ml) and released into medium containing 100 mM hydroxyurea (HU) for arrest in early S-phase. For arrest in metaphase, cells were released from M-NM-1-factor or HU arrest by filtration and re-suspension in YP medium supplemented with 5 mM methionine to deplete Cdc20. To inactivate cohesin loading, cells harbouring the temperature sensitive scc2-4 allele (Ciosk et al. 2000) were shifted to the restrictive temperature of 35M-BM-0C for 1 hour before release. To prevent metaphase spindle assembly, or to disassemble already formed spindles, 7.5 M-NM-<g/ml nocodazole was added to the medium for 1 hour. To wash out nocodazole, cells were filtered again, washed, and re-suspended in fresh medium lacking nocodazole for 1 hour. Expression of Scc1 to induce differentially epitope-tagged cohesin under control of the GAL1 promoter, or of Cdc14, was achieved by addition of 2% galactose for 1 hour to cultures grown in raffinose-containing medium. EXPERIMENTAL DESIGN Experiment type: ChIP-chip = Chromatin immunoprecipitation (ChIP) followed by hybridization to a high-density oligonucleotide microarray (chip). Number of hybridizations performed: 13 ChIP-chip samples; 2 SUPernatant samples Analysis: ChIP samples were normalized against SUPernatant fractions Quality control: Confirmation of results by duplication of experiments, by usage of different epitope tags for the same protein, and by usage of different subunits of the same protein complex. Also, whole cell extract, SUPernatant, and ChIP fractions were checked by Western blotting. Protocols: ChIP-chip and hybridization protocols were performed as previously described (Katou et al. 2003; Lengronne et al. 2004; Lengronne et al. 2006). A summary is provided below: ChIP. Cells were grown under the conditions described above and cross-linked with 1% formaldehyde. Cells were disrupted using a Multi-Beads Shocker, and whole cell extracts were sonicated to obtain 400-600 bp genomic DNA fragments. Chromatin immunoprecipitation (ChIP) was performed with anti-HA antibody or anti-PK monoclonal antibody coupled to protein A Dynabeads. Immunprecipates were eluted and incubated overnight at 65M-BM-:C to reverse the cross-link. The immunoprecipitated genomic DNA was then incubated with proteinase K, extracted twice with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RNaseA. The DNA was purified, concentrated by ethanol precipitation and amplified by PCR after random priming. 10ug of the amplified DNA was digested with DNase I and end-labelled using TdT Terminal Transferase and Biotin-N6-ddATP. Hybridization and scanning. Samples were hybridized in buffer containing SSPE, TritonX-100, denatured salmon sperm DNA and biotin-labelled control oligonucleotide, oligo B2. Samples were denatured at 95M-BM-:C for 10 minutes before being hybridized to the arrays for 16 hours at 42M-BM-:C in a GeneChip Hybridization Oven 640. The washing and scanning protocol (FlexMidi_euk2v3_450), provided by Affymetrix, was performed automatically on a GeneChip Fluidics Station 450. Arrays were scanned using the GeneChip Scanner 3000 7G, and primary analysis of tiling chip data was performed using the Affymetrix GeneChip Operating Software (GCOS). ARRAY DESIGN General array design: in situ synthesized arrays from Affymetrix Location and ID of each spot on arrays: available upon request from Affymetrix Probe type: oligonucleotide Availability of arrays: commercially available from Affymetrix S. cerevisiae (Chromosome VI) rikDACF, P/N# 510636 S. cerevisiae T iling Array (Forward), P/N# 520286 REFERENCES Ciosk R, Shirayama M, Shevchenko A, Tanaka T, Toth A, Shevchenko A, Nasmyth K (2000) Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol Cell 5: 1-20 Katou Y, Kanoh Y, Bandoh M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424: 1078-1083 Lengronne A, Katou Y, Mori S, Yokobayashi S, Kelly GP, Itoh T, Watanabe Y, Shirahige K, Uhlmann F (2004) Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 430: 573-578 Lengronne A, McIntyre J, Katou Y, Kanoh Y, Hopfner K-P, Shirahige K, Uhlmann F (2006) Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell 23: 787-799 Uhlmann F, Wernic D, Poupart M-A, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103: 375-386
2007-09-29 | E-GEOD-8661 | biostudies-arrayexpress
Project description:R-loops are transcription by-products that may constitute a threat to genome integrity. In addition to specific enzymes to remove them, eukaryotes rely on a number of mRNP biogenesis factors such as the THO complex, to prevent co-transcriptional R-loop formation. We show in Saccharomyces cerevisiae that R-loops are tightly and specifically linked with histone H3-Ser10 phosphorylation (H3S10P), a mark of chromatin condensation. Importantly, ChIP-chip analyses reveal a clear H3S10P accumulation at the pericentromeric chromatin during the G1-phase of the cell cycle only in R loop-accumulating yeast strains but not in those non-accumulating R-loops, and a significantly higher accumulation during S-phase. Such a difference can also be detected in a number of genes along the genome. ChIP-chip studies were perfomed with antibodies against Histone H3 and the phosphorylated Histone H3 at Serine10 in the yeast S. cerevisiae.
2013-11-13 | E-GEOD-46627 | biostudies-arrayexpress
Project description:R-loops are transcription by-products that may constitute a threat to genome integrity.We previously show in Saccharomyces cerevisiae that R-loops are tightly and specifically linked with histone H3-Ser10 phosphorylation (H3S10P), a mark of chromatin condensation. Here we analyse the hpr1-101 mutant. A point mutation that impairs transcription and mRNP biogenesis without increasing recombination. Importantly, ChIP-chip analyses reveal a clear H3S10P does not accumulate at the pericentromeric chromatin during the G1-phase of the cell cycle but during S-phase in non-accumulating R-loop hpr1-101. ChIP-chip studies were perfomed with antibodies against Histone H3 and the phosphorylated Histone H3 at Serine10 in the yeast S. cerevisiae.
2017-05-01 | E-GEOD-50110 | biostudies-arrayexpress
Project description:Higher-order chromosome structure is assumed to control various DNA-templated reactions in eukaryotes. Meiotic chromosomes implement developed structures called M-bM-^@M-^\axesM-bM-^@M-^] and M-bM-^@M-^\loopsM-bM-^@M-^]; both are suggested to tether each other, activating Spo11 to catalyze meiotic DNA double-strand breaks (DSBs) at recombination hotspots. We found that the Schizosaccharomyces pombe Spo11 homolog Rec12 and its partners form two distinct subcomplexes, DSBC (Rec6-Rec12-Rec14) and SFT (Rec7-Rec15-Rec24). Additionally, Mde2, whose expression is strictly regulated by the replication checkpoint, interacts with a component of each subcomplex. The SFT subcomplex binds to both axes via direct interaction of Rec15 with Rec10 in axes and DSB sites, hence axial Rec10 can partially tether DSB sites located in loops. Importantly, this multiprotein-based tethered axis-loop complex is destabilized in the absence of Mde2. We therefore propose a novel mechanism by which Mde2 functions as a recombination initiation mediator to tether axes and loops, in liaison with the meiotic replication checkpoint. ChIP-chip analyses of Rec10 (in wild type), Mde2 (in wild type and rec15M-bM-^HM-^F), and Rec15 (in wild type, rec10M-bM-^HM-^F, rec24M-bM-^HM-^F and mde2M-bM-^HM-^F) at meiosis 4 hours.
2012-07-27 | E-GEOD-31846 | biostudies-arrayexpress
Project description:In this study, we have investigated the molecular basis of Shh signalling during development of the secondary palate and how CNCC patterning and fate is influenced by the Shh signalling network. Using a gain-of-function mouse model to activate Smoothened (R26SmoM2) signalling in the palatal mesenchyme (Osr2-IresCre), we demonstrate ectopic Hh-Smo signalling results in fully penetrant cleft palate, disrupted oral-nasal patterning and defective palatine bone formation. We show that a series of Fox transcription factors, including the novel direct target Foxl1, function downstream of Hh signalling in the secondary palate. Furthermore, we demonstrate that Wnt/BMP antagonists, in particular Sostdc1, are positively regulated by Hh signalling, concomitant with down-regulation of key regulators of osteogenesis and BMP signalling effectors. Microarray analysis was performed on excised palatal shelves from Osr2-IresCre+/- (wild-type) and Osr2-IresCre;Smo+/M2 (mutant) embryos at embryonic day (E)13.5. Osr2-IresCre (PMID:17941042) and R26SmoM2 (PMID:15107405) mice have been described previously.
2018-10-02 | E-MTAB-5518 | biostudies-arrayexpress
Project description:Skeletal muscle atrophy is a debilitating condition associated with weakness, fatigue, and reduced functional capacity. Nuclear factor-kappaB (NF-κB) transcription factors play a critical role in atrophy. Knockout of genes encoding p50 or the NF-κB co-transactivator, Bcl-3, abolish disuse atrophy and thus they are NF-κB factors required for disuse atrophy. We do not know however, the genes targeted by NF-κB that produce the atrophied phenotype. Here we identify the genes required to produce disuse atrophy using gene expression profiling in wild type compared to Nfkb1 (gene encodes p50) and Bcl-3 deficient mice. There were 185 and 240 genes upregulated in wild type mice due to unloading, that were not upregulated in Nfkb1-/- and Bcl-3-/- mice, respectively, and so these genes were considered direct or indirect targets of p50 and Bcl-3. All of the p50 gene targets were contained in the Bcl-3 gene target list. Most genes were involved with protein degradation, signaling, translation, transcription, and transport. To identify direct targets of p50 and Bcl-3 we performed chromatin immunoprecipitation of selected genes previously shown to have roles in atrophy. Trim63 (MuRF1), Fbxo32 (MAFbx), Ubc, Ctsl, Runx1, Tnfrsf12a (Tweak receptor), and Cxcl10 (IP-10) showed increased Bcl-3 binding to κB sites in unloaded muscle and thus were direct targets of Bcl-3. p50 binding to the same sites on these genes either did not change or increased, supporting the idea of p50:Bcl-3 binding complexes. p65 binding to κB sites showed decreased or no binding to these genes with unloading. Fbxo9, Psma6, Psmc4, Psmg4, Foxo3, Ankrd1 (CARP), and Eif4ebp1 did not show changes in p65, p50, or Bcl-3 binding to κB sites, and so were considered indirect targets of p50 and Bcl-3. This work represents the first study to use a global approach to identify genes required to produce the atrophied phenotype with disuse. 24 mice were used based on 4 mice per group, 3 mouse genotypes (wild type, Nfkb1-/-, Bcl3-/-) and 2 conditions (weight-bearing and unloaded).
2011-01-10 | E-GEOD-23497 | biostudies-arrayexpress
Project description:Identification of sites of replication initiation by copy number analysis, comparing samples before and after entry into S phase. Experiments were performed in the following strains and conditions for mitotic cells: cdc25-22 (temperature-sensitive mutation arrests cells at G2/M for synchrony, otherwise wild type), nitrogen-rich conditions; wild type, haploid and diploid, nitrogen-depletion conditions. Experiments were performed in the following strains and conditions for meiotic cells: pat1-114 diploid (temperature-sensitive mutation induces a synchronous meiosis, otherwise wild type), nitrogen-depletion and nitrogen-rich conditions; pat1-114 diploid with altered Cdc45 levels, nitrogen-depletion conditions. Comparison of samples before and after S phase entry; dye-swap experiments were performed and averaged.
2014-02-20 | E-GEOD-53879 | biostudies-arrayexpress