Role of the Glc7 phosphatase regulatory subunit Ypi1 in progression through mitosis
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Antonio Casamayor
PROVIDER: E-GEOD-23134 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:The yeast proteins Pkh1 and Pkh2 are functional counterparts of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1). Cells carrying simultaneous deletion in both genes, PKH1 and PKH2 are not viable, at least in some genetic backgrounds (Casamayor et al., 1999; Inagaki et al., 1999). Consequently, a different strategy is needed to study the function of these apparently redundant gene products. The approach that has been successfully used to dissect the functions of these proteins consist in the use of a strain that combine the disruption of PKH2 with the temperature sensitive pkh1D398G allele (Inagaki et al., 1999). Since Pkh1 and Pkh2 phosphorylate Pkc1 in vitro in its activation loop (Thr983), and the cell lysis defects of the pkh1D398G pkh2 strain at the restrictive temperature are partially restored by constitutive activation of the Pkc1-Slt2 MAPK pathway, it has been suggested that the Pkh activity is important for maintenance of the cell wall integrity in S. cerevisiae (Inagaki et al., 1999; Friant et al., 2001). We considered it necessary that a different genetic strategy would be convenient in order to carry out specific studies about the functions of the Pkh proteins. In order to avoid incubation at temperatures that activate the Slt2 pathway, and induce cellular lysis, we decided to use a strategy based on the use of a regulatable promoter. We replaced 487 bp located immediately upstream the start PKH2 codon by a cassette containing the doxycycline-repressed promoter tetO7. This substitution is positioned in a 1.3 kbp intergenic region containing divergent promoters and it seems unlikely that may affect expression of the IZH4, the upstream gene, which encode a family of four paralogous membrane proteins involved in zinc ion homeostasis and whose expression is regulated by fatty acids and zinc altered levels. In this strain (MB002) the PKH2 expression could be switched off by addition of doxycycline. In order to deplete the cell for the Pkh activity, we disrupted both, the PKH1 and PKH1 coding regions, obtaining the new SDP8 strain. Our results show that the long term depletion of Pkh results in a cell transcriptional response that include overexpression of genes typically induced in response to oxidative stress, as well as up-regulation of chaperons and genes that induce the unfolded protein response. Four samples were analyzed: the SDP8 strain, a tetO7 conditional mutant, and its isogenic WT strain both, in the presence and in the absence of doxycyline (for 8 and 24 h). We compared the expression profile of: 1) SDP8 + Dox after 8h vs WT + Dox after 8h 2) SDP8 + Dox after 24h vs WT + Dox after 24h A Dye-swap was carried out for each comparison of samples. Total number of chips analyzed: 4.
2015-04-30 | E-GEOD-32623 | biostudies-arrayexpress
Project description:The yeast proteins Pkh1 and Pkh2 are functional counterparts of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1). Cells carrying simultaneous deletion in both genes, PKH1 and PKH2 are not viable, at least in some genetic backgrounds (Casamayor et al., 1999; Inagaki et al., 1999). Consequently, a different strategy is needed to study the function of these apparently redundant gene products. The approach that has been successfully used to dissect the functions of these proteins consist in the use of a strain that combine the disruption of PKH2 with the temperature sensitive pkh1D398G allele (Inagaki et al., 1999). Since Pkh1 and Pkh2 phosphorylate Pkc1 in vitro in its activation loop (Thr983), and the cell lysis defects of the pkh1D398G pkh2 strain at the restrictive temperature are partially restored by constitutive activation of the Pkc1-Slt2 MAPK pathway, it has been suggested that the Pkh activity is important for maintenance of the cell wall integrity in S. cerevisiae (Inagaki et al., 1999; Friant et al., 2001). We considered it necessary that a different genetic strategy would be convenient in order to carry out specific studies about the functions of the Pkh proteins. In order to avoid incubations at temperatures that activate the Slt2 pathway, and induce cellular lysis, we decided to use a strategy based on the use of a regulatable promoter. We replaced 487 bp located immediately upstream the start PKH2 codon by a cassette containing the doxycycline-repressed promoter tetO7. This substitution is positioned in a 1.3 kbp intergenic region containing divergent promoters and it seems unlikely that may affect expression of the IZH4, the upstream gene, which encode a family of four paralogous membrane proteins involved in zinc ion homeostasis and whose expression is regulated by fatty acids and zinc altered levels. In this strain (MB002) the PKH2 expression could be switched off by addition of doxycycline. In order to deplete the cell for the Pkh activity, we disrupted both, the PKH1 and PKH3 coding regions, obtaining the new SDP8 strain. Our results show that depletion of Pkh results in a differential transcriptional response in front of a heat shock stress. The general transcriptional response was attenuated in the SDP8 when compared when WT cells, but lack of Pkh activity did not affect the repression of the ribosomal proteins. Four samples were analyzed: the SDP8 strain, a tetO7 conditional mutant, and its isogenic WT strain both, in the presence and in the absence of doxycyline (for 8 and 24 h). We compared the expression profile of: 1) WT + 16h Dox + 40 min heat shock vs WT + 16h Dox. 2) SDP8 + 16h Dox + 40 min heat shock vs SDP8 + 16h Dox. A Dye-swap was carried out for each comparison of samples. Total number of chips analyzed: 4.
2015-04-30 | E-GEOD-33904 | biostudies-arrayexpress
Project description:The change of genome-wide transcription profiles as a result of Gis1 overexpression is monitored by comparing the transcriptomes isolated from cells where Gis1 overexpression is switched on or off. GIS1 was cloned into pCM190 vector under the control of the tetO7 promoter. The promoter is switched on when there is no doxycycline but off with doxycycline (20ug/ml). Cells were grown in medium with doxycycline, harvested, washes twice in sterile water, resuspended in the same medium with doxycycline (Dox+) or without doxycycline (dox-) and grown for additional 6 hours. Samples were taken for each condition at 3 and 6 hours. Time 0 sample was taken before resuspension.
2009-12-01 | E-MEXP-2354 | biostudies-arrayexpress
Project description:Regulation of gene expression programs is crucial for the survival of microbial pathogens in host environments and for their ability to cause disease. Here we investigated the epigenetic regulator RSC (Remodels the Structure of Chromatin) in the most prevalent human fungal pathogen Candida albicans. We addressed the global functions of RSC in C. albicans by analyzing the changes in the proteome of cells lacking the catalytic subunit Sth1 using DIA-MS (Data Independent Acquisition-Mass Spectrometry). For this purpose, we generated a conditional mutant harboring a doxycycline (dox)-repressible allele of STH1 (sth1∆/pTetO-STH1-MYC) and treated this mutant with dox for Sth1 depletion. To assess the effect of dox alone on gene expression, a near-isogenic sth1∆/STH1-MYC strain was also analysed.
2020-11-11 | PXD017990 | Pride
Project description:To investigate the impact of Rap1 binding on nearby gene expression during the transition from vegetative growth to meiosis, we measured RNA abundance in cells depleted (plus doxycycline) or not depleted (minus doxycycline) of Rap1. RNA abundance was measured in depleted and non-depleted condition in respiratory cells (pre-meiotic/YPA 20 hours) and in cells undergoing synchronous meiosis/sporulation in the budding yeast Saccharomyces cerevisiae. There is growing recognition that the binding of a transcription factor near a gene does not always indicate regulatory function, and further that a single factor may function to either activate or repress its targets depending on the cellular context. We examined these issues through a series of experiments involving the S. cerevisiae transcription factor Rap1, and its function throughout critical metabolic and developmental transitions between vegetative growth, respiratory growth, meiosis and sporulation. We simultaneously monitored the expression of all genes and the genomic binding locations of Rap1 throughout the timecourse. Genes downstream of Rap1 binding were activated and repressed dynamically, but a change - or lack of change - in Rap1 binding status was not predictive of activation, repression, or no change in regulation. Despite this, we show that Rap1 is required, at a given point in time, for both activation and repression of different gene targets, within the same cell. Specification of the transcriptional consequences of Rap1 binding is thus highly promoter-specific. The presence of other transcription factor binding motifs, the subtype of Rap1 motif, and the underlying chromatin structure of the promoter cannot fully account for the observed transcriptional outcomes. There is a better accounting for the dynamic binding behavior of Rap1 including specification of an expanded meiotic target set through a Tup1- dependent nucleosome-loss mechanism. The variable and dynamic association between binding and transcription in this simple unicellular system portends a similarly volatile relationship in more complex eukaryotes. Biological interpretations of transcription factor occupancy should be made cautiously and in conjunction with supporting data obtained under the precise condition of interest. SK1 yeast strain SHy20 (MATa/MATalpha lys2/lys2 ura3/ura3 ho::LYS2/ho::LYS2 leu2::hisG/leu2::hisG his4x/his4b URA3::CMV-tetTA/URA3::CMV-tetTA RAP1promoter::kanR-tetO7-TATA/RAP1promoter::kanR-tetO7-TATA). An overnight culture of YPD was used to innoculate a YPA culture (minus doxycycline; Rap1 on). The YPA culture was grown for 8 hours and then split into minus doxycyline (Rap1 on) and plus doxycylcine (Rap1 depleted) samples. Minus and plus doxycyline samples were collected after an additional 12 hours in YPA (respiratory growth/pre-meiotic; YPA 20 hours) and 0 hr, 3hr, 6hr, 9hr and 12hr after transfer to sporulation media (SM) with or without doxycycline. 3 separate biological replicates were used for each condition/time-point. Labeled total RNA from Rap1 depeleted and non-depleted samples were competitively hybridized to yeast whole genome PCR based spotted arrays (resolution ~1kb). The Rap1-depletion ChIP and RNA abundance/expression analysis were carried out on the same biological samples. The three replicates of Rap1-depletion RNA abundance/expression analysis correspond to replicates 2, 3, and 5 of the Rap1-depletion Rap1 ChIP experiment.
2016-09-01 | E-GEOD-75259 | biostudies-arrayexpress
Project description:Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, in addition to DNA repair, genome stability is also controlled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can alter the DNA damage cell cycle checkpoints. Here, we show that hypoxia (24h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, flow cytometric bar-coding analysis of individual cells showed that the levels of several G2 checkpoint regulators were reduced in G2 phase cells after hypoxic exposure, in particular cyclin B1. These effects were accompanied by decreased Cyclin dependent kinase (CDK) activity in G2 phase cells after hypoxia. Furthermore, cells pre-exposed to hypoxia showed a longer G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (~0.03 % O2 20h and 0.2% O2 72h). These results demonstrate that the DNA damage G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors. We measured gene expression changes in U2OS cells after treatment with 0.2% hypoxia for 24 hours. The data were used in the exploration of hyopxia induced alterations in the G2 checkpoint.
2016-01-04 | E-GEOD-70530 | biostudies-arrayexpress
Project description:Mre11, together with Rad50 and Xrs2/NBS, plays pivotal roles in homologous recombination, repair of DNA double strand breaks (DSBs), activation of damage-induced checkpoint, and telomere maintenance. Using DNA microarray assays to analyze yeast mutants (mre11delta, rad50delta, and spo11Y135F) defective for meiotic DSB formation, we demonstrate that the absence of Mre11 in yeast causes specific effects on regulation of a class of meiotic genes for spore development. The transcriptional deficiency was not observed in other DSB mutants such as rad50delta and spo11Y135F, suggesting the transcriptional defect in mre11delta is due to neither lack of meiotic DSB formation, nor disintegrity of Mre11-Rad50-Xrs2 complex.These defects were confirmed by northern and lacZ reporter gene assays. Experiment Overall Design: Gene expression data from wild type, mre11delta, rad50delta, and spo11Y135F cells in premeiosis (meiosis 0 hr) and prophase (meiosis 4 hr). Affymetrix GeneChip YG-S98 was used. All strains used were SK1 background diploid cells.
2007-10-23 | E-GEOD-6620 | biostudies-arrayexpress
Project description:Here we exploit the essential process of XM-bM-^@M-^Pchromosome dosage compensation to elucidate basic mechanisms that control the assembly, genomeM-bM-^@M-^Pwide binding, and function of gene regulatory complexes that act over large chromosomal territories. We demonstrate that a subunit of C. elegans MLL/COMPASS, a gene-activation complex, acts within the dosage compensation complex (DCC), a condensin complex, to target the DCC to both X chromosomes of hermaphrodites and thereby reduce chromosome-wide gene expression. The DCC binds to two categories of sites on X: rex sites that recruit the DCC in an autonomous, sequence- dependent manner, and dox sites that reside primarily in promoters of expressed genes and bind the DCC robustly only when attached to X. We find that DCC mutants that abolish rex-site binding do not eliminate dox-site binding, but instead reduce it to the level observed at autosomal binding sites in wild-type animals. Changes in DCC binding to these non-rex sites occur throughout development and correlate with transcriptional activity of adjacent genes. Moreover, autosomal DCC binding is enhanced by rex-site binding in cis in X-autosome fusion chromosomes. Thus, dox and autosomal sites exhibit similar binding properties. Our data support a model for DCC binding in which low-level DCC binding at dox and autosomal sites is dictated by intrinsic properties correlated with high transcriptional activity. Sex-specific DCC recruitment to rex sites then greatly elevates DCC binding to dox sites in cis, which lack intrinsically high DCC affinity on their own. We also show here that the C. elegans DCC achieves dosage compensation through its effects on transcription. Total RNA was extracted from fed L1 worms in order to compare total RNA levels with that of mixed embryos.
2011-03-01 | E-GEOD-25831 | biostudies-arrayexpress
Project description:Background: thyroid transcription factor-1 (TTF-1) is known to play key roles in thyroid organogenesis, in thyroid cell proliferation and in the expression of genes involved in thyroid differentiated function. Objective: to understand why many human thyroid cancer cell lines keep producing TTF-1 despite the loss of differentiation. Methods: a chimeric protein acting as a functional antagonist of TTF-1 transcriptional activity was expressed conditionally in 8505C cells originating from an undifferentiated thyroid carcinoma. The consequences of the inhibition of TTF-1 activity in the cells expressing the antagonist on cell proliferation, mRNA and miRNA expression were analyzed. Results: we observed a growth arrest of 8505C thyroid cancer cells when the endogenous TTF-1 transcriptional activity was inhibited. It correlated with decreased levels of several mRNAs encoding positive effectors of cell proliferation like CDK1 and cyclinB1, and increased levels of various mRNAs encoding negative regulators of cell division like CDKN2B and DUSP6. By contrast, no significant change was observed in the miRNA population. Conclusion: the persistence of TTF-1 expression observed in most dedifferentiated human thyroid cancer cell lines is likely to be explained by the fact that TTF-1 activity is still required for proliferation of these tumor cells as demonstrated here in the 8505C cell line. Microarrays miRNA hybridizations were performed on 8505c cells expressing a TTF1 antagonist (Engr HD âDox) versus non-expressing cells (Engr HD +Dox), on cells expressing the control protein (Engr HDm â Dox) versus non-expressing cells (Engr HDm + Dox), and on cell expressing the TTF1 antagonist (Engr HDâ Dox) versus the cells expressing the control protein (Engr HDmâ Dox). Hybridizations were replicated with dye swap.
2016-06-01 | E-GEOD-58243 | biostudies-arrayexpress
Project description:Defects in DNA damage responses may underlie genetic instability and malignant progression in melanoma. Cultures of normal human melanocytes (NHMs) and melanoma lines were analyzed to determine whether global patterns of gene expression could predict the efficacy of DNA damage cell cycle checkpoints that arrest growth and suppress genetic instability. NHMs displayed effective G1 and G2 checkpoint responses to ionizing radiation-induced DNA damage. A majority of melanoma cell lines (11/16) displayed significant quantitative defects in one or both checkpoints. Melanomas with B-RAF mutations as a class displayed a significant defect in DNA damage G2 checkpoint function. In contrast the epithelial-like subtype of melanomas with wildtype N-RAS and B-RAF alleles displayed an effective G2 checkpoint but a significant defect in G1 checkpoint function. RNA expression profiling revealed that melanoma lines with defects in the DNA damage G1 checkpoint displayed reduced expression of p53 transcriptional targets, such as CDKN1A and DDB2, and enhanced expression of proliferation-associated genes, such as CDC7 and GEMININ. A Bayesian analysis tool was more accurate than significance analysis of microarrays for predicting checkpoint function using a leave-one-out method. The results suggest that defects in DNA damage checkpoints may be recognized in melanomas through analysis of gene expression. Experiment Overall Design: Normal human melanocyte and melanoma cell lines w/wo mutations in B-raf or N-ras were treated with 1.5 Gy IR irridiartion for G1 and G2 checkpoint determination. RNA was isolated from exponentially growing cultures and applied for microarray hybridizaton with Agilent 44 K (G4112A) array.
2008-06-15 | E-GEOD-7469 | biostudies-arrayexpress