Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Jhd2 S321A S340A mutant Transcriptomes
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE175870 | GEO | 2022/08/29
REPOSITORIES: GEO
Similar Datasets
Project description:Cells need to coordinate gene expression with their metabolic states to maintain cell homeostasis and growth. However, how cells transduce nutrient availability to appropriate gene expression response via histone modifications remains poorly understood. Here, we report that glycolysis promotes H3K4me3 by activating Tpk2, the catalytic subunit of protein kinase A (PKA) via the Ras-cyclic AMP (cAMP) pathway. Further study showed that Tpk2 antagonizes Jhd2-catalyzed H3K4 demethylation by phosphorylating Jhd2 at S321 and S340 in response to glucose availability.Mechanistically, Tpk2-catalyzed Jhd2 phosphorylation inhibits its overall binding to chromatin and promotes its polyubiquitination by the E3 ubiquitin ligase Not4 and degradation by the proteasome. In addition, Tpk2-catalyzed Jhd2 phosphorylation also maintains H3K14ac by preventing the binding of Rpd3 to chromatin. By inhibiting the activity of Jhd2 and Rpd3, Tpk2-catalyzed Jhd2 phosphorylation regulates gene expression and promotes autophagy. Thus, regulation of Jhd2 by the Ras-cAMP-PKA pathway shed lights on how cells rewire their biological responses to glucose availability.
2022-08-29 | GSE175868 | GEO
Project description:This work uncovers a novel and biologically significant mechanism that directly connects nutrient availability to histone modifications and gene transcription. We report that glycolysis promotes histone H3K4 trimethylation (H3K4me3) by activating Tpk2, a catalytic subunit of protein kinase A (PKA) via the Ras-cyclic AMP (cAMP) pathway. Glucose-activated PKA (Tpk2) inhibits Jhd2-catalyzed H3K4 demethylation by phosphorylating Jhd2 at serine 321 and serine 340. In addition, Tpk2- catalyzed Jhd2 phosphorylation promotes H3K14ac by preventing the binding of Rpd3 at chromatin.
2022-08-29 | PXD030815 | Pride
Project description:The pattern of gene transcription in Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. To investigate the requirement of PKA for glucose control of gene expression, we have analyzed global transcription in strains devoid of PKA activity. In S. cerevisiae three genes, TPK1, TPK2, TPK3, encode catalytic subunits of PKA and the triple mutant tpk1 tpk2 tpk3 is unviable. We have worked, therefore, with two strains, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4, that bear suppressor mutations,. We have identified different classes of genes that can be induced, or repressed, by glucose in the absence of PKA. Among these genes, some are also controlled by a redundant signalling pathway involving PKA activation, while others do not respond to an increase in cAMP concentration. On the other hand, among genes which do not respond to glucose in the absence of PKA, some show a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways.
2011-08-09 | GSE27541 | GEO
Project description:The conserved cAMP-dependent protein kinase (PKA) holoenzyme is composed of two catalytic and two regulatory subunits. It plays critical roles in the regulation of many biological processes in eukaryotic organisms. In the human fungal pathogen Candida albicans, the PKA kinase has been extensively investigated for its importance in the regulation of morphological transitions and virulence. It has been long thought that the PKA catalytic subunit is essential for cell viability in C. albicans. Paradoxically, the single adenylyl cyclase-encoding gene, CRY1, which is required for the production of cAMP in C. albicans, is not essential for cell growth. In this study, we successfully generated a null double mutant of TPK1 and TPK2 (tpk2/tpk2 tpk1/tpk1 or t2t1), which encode two isoforms of the PKA catalytic subunit in C. albicans. We reevaluated the roles of the PKA catalytic subunit in cell growth and phenotypic transitions. Inactivation of the PKA catalytic subunit by deletion of both TPK1 and TPK2 blocked filamentation and dramatically attenuated the ability of white-to-opaque switching, but promoted sexual mating in C. albicans. Tpk2 plays a major role in these regulations, while Tpk1 generally functions as a negative regulator in morphological transitions and sexual mating. A comparative transcriptomic analysis demonstrated that the t2t1 and cyr1/cyr1 mutants exhibited similar global gene expression profiles. Compared to the WT strain, the general transcriptional activity and expression of genes involved in metabolism, translation, biosynthesis, adhesion and filamentation are significantly decreased in both the t2t1 and cyr1/cyr1 mutants. And a portion of stress-response and cell wall-related genes were upregulated in these mutants, which is consistent with their increased ability of anti-stresses. To further explore the global regulatory role of the PKA kinase, we performed quantitative phosphoproteomics analysis. Combining with bioinformatics analyses, we identified 181 potential PKA phosphorylation targets, which represent 148 unique proteins involved in a wide spectrum of biological processes. Cell wall and membrane-related proteins (e.g. Ecm3, Bni1, and Smi1) were enriched in Tpk1-specific targets, while Tpk2-specific substrates include transporters, filamentation and cytoskeleton-related proteins (e.g. Smf3, Sep7, and Mhp1). There were also many Tpk1 and Tpk2 overlapped and coordinately regulated-substrates. Our study clarifies the essentiality of the PKA catalytic subunit and shed new insights into the global regulatory features of the cAMP/PKA pathway in C. ablicans. The t2t1 null mutant generated in this study would also be a new resource for the field to study this important pathway.
2017-08-18 | GSE92621 | GEO
Project description:The pattern of gene transcription in Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. To investigate the requirement of PKA for glucose control of gene expression, we have analyzed global transcription in strains devoid of PKA activity. In S. cerevisiae three genes, TPK1, TPK2, TPK3, encode catalytic subunits of PKA and the triple mutant tpk1 tpk2 tpk3 is unviable. We have worked, therefore, with two strains, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4, that bear suppressor mutations,. We have identified different classes of genes that can be induced, or repressed, by glucose in the absence of PKA. Among these genes, some are also controlled by a redundant signalling pathway involving PKA activation, while others do not respond to an increase in cAMP concentration. On the other hand, among genes which do not respond to glucose in the absence of PKA, some show a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. The goal of the present study was to investigate the occurrence of PKA-independent glucose signalling in S. cerevisiae. To this end, we have used global transcription analysis to study the effects of glucose on yeast strains completely devoid of PKA activity. In S. cerevisiae three genes TPK1, TPK2,and TPK3 encode catalytic subunits of PKA. While strains expressing only one of these genes grow normally, a triple null mutant (tpk1 tpk2 tpk3) is not viable (Toda et al 1987). Identification of different mutations able to suppress the growth defect of the triple mutant (Garrett and Broach 1989, Reinders et al 1998, Smith et al 1998) has allowed to determine what is the crucial function of PKA. As shown in Fig.1, PKA is needed to counteract the negative effect of the protein kinase Yak1 on yeast growth (Hartley et al 1994, Moriya et al 2001). In the presence of PKA the protein kinase Rim15 (Reinders et al 1998) and the transcription factors Msn2 and Msn4 (Görner et al 1998) can be phosphorylated and exported to the cytoplasm, transcription of the YAK1 gene, which is activated by Msn2/Msn4 (Smith et al 1998), is reduced, Yak1 levels remain low and growth is not hindered. In the absence of PKA, Rim15 remains in the nucleus where it can activate Msn2/Msn4 (Cameroni et al 2004) that turn on YAK1 transcription, thus blocking growth. This explains why strains lacking Rim15, Msn2/Msn4 or Yak1 no longer require PKA for growth. In this work we have used two isogenic strains lacking PKA and carrying the suppressor mutations msn2 msn4 or yak1. Two different suppressor mutants were used with the aim to enable a dissection of effects of the lack of PKA and effects of the suppressor mutations themselves.
2011-08-08 | E-GEOD-27541 | biostudies-arrayexpress
Project description:To assess the relative contributions of the PKA and MAPK pathways in Ras stimulation of filamentation, we examined the change in transcriptional profile following activation of Ras in strains in which one of these two pathways has been severed. These arrays contain the experiments involving WT strain with glucose addition, Ras2G19V strain with galactose induction, tpk2-w strain as a control, and RasG19V activation in the tpk2-w background. Activation of Ras in the Σ1278b background – by galactose addition to a gal1 PGAL10-RAS2G19V strain - causes a massive restructuring of the transcription pattern of the cell, which closely resembles the changes induced by addition of glucose. To assess the extent to which these Ras-induced changes depended on signaling through PKA, we compared the expression profile following induction of activated Ras in a wild type background to that following induction of activated Ras in the bcy1 tpk1 tpk3 tpk2V218G. Keywords: time course
2007-08-18 | GSE8805 | GEO
Project description:Besozzi2012 - Oscillatory regimes in the Ras/cAMP/PKA pathway in S.cerevisiae
Mechanistic model of the Ras/cAMP/PKA in yeast S.cerevisiae. The Ras/cAMP/PKA pathway plays a major role in the regulation of metabolism, stress resistance and cell cycle progress and is tightly regulated by multiple feedback loops, exerted by the protein kinase A (PKA). This model investigates the dynamics of the second messenger cAMP on Ras/cAMP/PKA pathway, to determine the effects of the feedback mechanisms on establising stable oscillatory regimes.
The model has been defined according to the stochastic formulation of chemical kinetics [Gillespie DT, 1977]
, which requires to specify the set of molecular species occurring in the pathway and their respective interactions, formally described as a set of biochemical reactions.
The volume considered for this system is 30fL; this value can be used to convert the model into the deterministic formulation.
This model is described in the article:
The role of feedback control mechanisms on the establishment of oscillatory regimes in the Ras/cAMP/PKA pathway in S. cerevisiae.
Besozzi D, Cazzaniga P, Pescini D, Mauri G, Colombo S, Martegani E.
EURASIP J Bioinform Syst Biol. 2012 Jul 20;2012(1):10.
Abstract:
In the yeast Saccharomyces cerevisiae, the Ras/cAMP/PKA pathway is involved in the regulation of cell growth and proliferation in response to nutritional sensing and stress conditions. The pathway is tightly regulated by multiple feedback loops, exerted by the protein kinase A (PKA) on a few pivotal components of the pathway. In this article, we investigate the dynamics of the second messenger cAMP by performing stochastic simulations and parameter sweep analysis of a mechanistic model of the Ras/cAMP/PKA pathway, to determine the effects that the modulation of these feedback mechanisms has on the establishment of stable oscillatory regimes. In particular, we start by studying the role of phosphodiesterases, the enzymes that catalyze the degradation of cAMP, which represent the major negative feedback in this pathway. Then, we show the results on cAMP oscillations when perturbing the amount of protein Cdc25 coupled with the alteration of the intracellular ratio of the guanine nucleotides (GTP/GDP), which are known to regulate the switch of the GTPase Ras protein. This multi-level regulation of the amplitude and frequency of oscillations in the Ras/cAMP/PKA pathway might act as a fine tuning mechanism for the downstream targets of PKA, as also recently evidenced by some experimental investigations on the nucleocytoplasmic shuttling of the transcription factor Msn2 in yeast cells.
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2024-09-02 | BIOMD0000000478 | BioModels
Project description:To assess the relative contributions of the PKA and MAPK pathways in Ras stimulation of filamentation, we examined the change in transcriptional profile following activation of Ras in strains in which one of these two pathways has been severed. These arrays contain the experiments involving WT strain with glucose addition, Ras2G19V strain with galactose induction, tpk2-w strain as a control, and RasG19V activation in the tpk2-w background. Activation of Ras in the Σ1278b background – by galactose addition to a gal1 PGAL10-RAS2G19V strain - causes a massive restructuring of the transcription pattern of the cell, which closely resembles the changes induced by addition of glucose. To assess the extent to which these Ras-induced changes depended on signaling through PKA, we compared the expression profile following induction of activated Ras in a wild type background to that following induction of activated Ras in the bcy1 tpk1 tpk3 tpk2V218G. Keywords: time course RNA from strains grown as described was extracted using the RNeasy Mini Kit (Qiagen, California, USA) with mechanical disruption. RNA labeling and hybridization to Agilent oligonucleotide microarrays were done according to manufacturer’s instructions (Agilent Technologies, California, USA). RNA and cRNA concentrations were measured with NanoDrop (NanoDrop Technologies, Delaware, USA). 250ng labeled cRNA was used for each hybridization. The zero minute time point samples were labeled with Cy3 and others with Cy5. At least two technical replicates of each hybridization were performed. Hybridized microarrays were scanned with Agilent Microarray Scanner G2505B and the images were prepared for data analysis with Feature Extraction Software version 7.5 (Agilent Technologies, California, USA). Data were filtered and analyzed using PUMAdb based on the Stanford Microarray Database package.
2007-08-18 | E-GEOD-8805 | biostudies-arrayexpress
Project description:The second messenger cAMP acts via protein kinase A (PKA) to induce apoptosis by mechanisms that are poorly understood. Here, we assessed a role for mitochondria and analyzed gene expression in cAMP/PKA-promoted apoptosis by comparing wild-type (WT) S49 lymphoma cells and the S49 variant, D- (cAMP-deathless), which lacks cAMP-promoted apoptosis but has wild-type levels of PKA activity and cAMP-promoted G1 growth arrest. Treatment of WT, but not D-, S49 cells with 8-CPT-cAMP for 24 h induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and Smac and increase in caspase-3 activity. Gene expression analysis (using Affymetrix 430 2.0 Arrays) revealed that WT and D- cells incubated with 8-CPT-cAMP have similar, but non-identical, extents of cAMP-regulated gene expression at 2h (~800 transcripts) and 6h (~1000 transcripts) (|Fold|>2, P<0.06); by contrast, at 24h ~2500 and ~1100 transcripts were changed in WT and D- cells, respectively. Using an approach that combined regression analysis, clustering and functional annotation to identify transcripts that showed differential expression between WT and D- cells, we found differences in cAMP-mediated regulation of mRNAs involved in transcriptional repression, apoptosis, the cell cycle, RNA splicing, Golgi and lysosomes. The 2 cell lines differed in CREB phosphorylation and expression of the transcriptional inhibitor Icer and in cAMP-regulated expression of genes in the Inhibitor of apoptosis (IAP) and Bcl families. The findings indicate that cAMP/PKA-promoted apoptosis of lymphoid cells occurs via mitochondrial-mediated events and imply that such apoptosis involves gene networks in multiple biochemical pathways. Experiment Overall Design: S49 D- cells were treated with 8-CPT-cAMP over the course of 24 hours. Cells were prepared for hybridization to microarrays at times 0-untreated, 2h, 6h, and 24h after treatment with 8-CPT-cAMP. Experimental replicates were as follows n=4 for time 0 and n=3 for 2, 6, and 24h post treatment.
2008-06-16 | E-GEOD-9727 | biostudies-arrayexpress
Project description:Hormones and nutrients often induce genetic programs via signaling pathways that interface with gene-specific activators. Activation of the cAMP pathway, for example, stimulates cellular gene expression by means of the PKA-mediated phosphorylation of cAMP-response element binding protein (CREB) at Ser-133. Here, we use genome-wide approaches to characterize target genes that are regulated by CREB in different cellular contexts. CREB was found to occupy approximately 4,000 promoter sites in vivo, depending on the presence and methylation state of consensus cAMP response elements near the promoter. The profiles for CREB occupancy were very similar in different human tissues, and exposure to a cAMP agonist stimulated CREB phosphorylation over a majority of these sites. Only a small proportion of CREB target genes was induced by cAMP in any cell type, however, due in part to the preferential recruitment of the coactivator CREB-binding protein to those promoters. These results indicate that CREB phosphorylation alone is not a reliable predictor of target gene activation and that additional CREB regulatory partners are required for recruitment of the transcriptional apparatus to the promoter.
2006-05-09 | E-WMIT-11 | biostudies-arrayexpress