ABSTRACT: GRO (Genomic run-on) experiments with different mutants that affect to the accumulation of non active RNA pol II along the yeast genome. Keywords: Genomic run-on GRO Overall design: There are 4 different strains: rap1-sil (without the silencing domain), RAP1(both from Graham I.R. et al 1999) and tpk1 & tpk2 mutants (Euroscarf). For each experiment there are GRO data (Transcription Rate) and gDNA data used for normalizing the GRO signals. The last number of the GRO filters correspond to the number of gDNA filters.There are 3 independent biological replicates of Rap1 and rap1-sil experiments and 2 for the tpk1 & tpk2 experiments.
Project description:GRO (Genomic run-on) experiments with different mutants that affect to the accumulation of non active RNA pol II along the yeast genome. Keywords: Genomic run-on GRO There are 4 different strains: rap1-sil (without the silencing domain), RAP1(both from Graham I.R. et al 1999) and tpk1 & tpk2 mutants (Euroscarf). For each experiment there are GRO data (Transcription Rate) and gDNA data used for normalizing the GRO signals. The last number of the GRO filters correspond to the number of gDNA filters.There are 3 independent biological replicates of Rap1 and rap1-sil experiments and 2 for the tpk1 & tpk2 experiments.
Project description:RPCC (RNA pol II ChIP-on-chip) experiments with different mutants that affect to the accumulation of non active RNA pol II along the yeast genome. Keywords: ChIP-chip Overall design: There are 4 different strains: rap1-sil (without the silencing domain), RAP1 (both from Graham I.R. et al 1999) and tpk1 & tpk2 mutants (Euroscarf). The IP was done using an Ab against the RNApol II CTD (8WG16 Covance).There are 3 independent biological replicates of Rap1 and rap1-sil experiments and 2 for the tpk1 & tpk2 experiments.
Project description:RPCC (RNA pol II ChIP-on-chip) experiments with different mutants that affect to the accumulation of non active RNA pol II along the yeast genome. Keywords: ChIP-chip There are 4 different strains: rap1-sil (without the silencing domain), RAP1 (both from Graham I.R. et al 1999) and tpk1 & tpk2 mutants (Euroscarf). The IP was done using an Ab against the RNApol II CTD (8WG16 Covance).There are 3 independent biological replicates of Rap1 and rap1-sil experiments and 2 for the tpk1 & tpk2 experiments.
Project description:Analysis of genome-wide differences of transcription using Genomic run-on (GRO), RNApol II ChIP-on-Chip, cDNA analysis and ChIP-on-Chip. This SuperSeries is composed of the following subset Series: GSE14060 RNA pol II ChIP on chip (RPCC) GSE14077 RPCC analysis of rap1-sil, tpk1 & tpk2 GSE14080 GRO analysis of rap1-sil, tpk1 & tpk2 GSE14082 Analysis of Spt16 depletion GSE1002 YPD to YPGal timecourse Refer to individual Series
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. Overall design: total RNA profiles of wild type,tpk2/tpk2 tpk1/tpk1 double mutant, and cyr1/cyr1 mutant
Project description:To confirm that Rap1-depletion resulted in decreased binding of Rap1 to its target sites, we performed compared Rap1 ChIP-chip experiments in respiratory cells (pre-meiotic/YPA 20 hours) depleted (plus doxycycline for 12 hours) or not depleted (minus doxycycline) of Rap1. 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). 6 separate biological replicates were used. Labeled ChIP DNA 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.
Project description:The cyclic AMP-protein kinase A pathway has a central role in the biology of Candida albicans, a prominent fungal pathogen of humans. The two catalytic subunits for cyclic AMP-dependent protein kinase, Tpk1 (orf19.4892) and Tpk2 (orf19.2277), have divergent roles, and most studies indicate a more pronounced role for Tpk2. Here we dissect two Tpk1-responsive properties: adherence and cell wall integrity. Homozygous tpk1/tpk1 mutants are hyperadherent, and a Tpk1 defect enables biofilm formation in the absence of Bcr1, a central transcriptional regulator of biofilm adhesins. Microarray analysis revealed an enrichment for cell wall and surface functions among Tpk1-repressed genes, and overexpression of individual target genes indicates that cell surface proteins Als1, Als2, Als4, Csh1, and Csp37 contribute to Tpk1-regulated adherence. Tpk1 is also required for cell wall integrity, but has no role in the cell wall integrity gene expression response. Interestingly, increased expression of the adhesin gene ALS2 conferred a cell wall defect, as manifested in hypersensitivity to the cell wall inhibitor caspofungin and a shallow cell wall structure. Our findings indicate that Tpk1 has a central role in C. albicans cell wall properties that is exerted through repression of select cell surface protein genes. Two-color microarrays using a closed-loop experimental design to determine the effects of a ∆tpk1 deletion in the absence or presence of the antifungal Caspofungin (CF)
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. Overall design: 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.