Prevorovsky, Martin*
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Transcription factors are prominent regulators of gene expression that execute responses to various intracellular and extracellular stimuli. Recombinant transcription reporter systems can be conveniently used to study the DNA binding preferences and regulatory activity of a transcription factor under a range of conditions. Several reporter genes have been used to study transcription regulation in the fission yeast Schizosaccharomyces pombe. Each of these reporters has distinct advantages, such as high sensitivity or ease of use, and limitations, such as prohibitive costs or use of hazardous substances. To combine the strengths and mitigate the weaknesses of individual reporter genes, we have created pREPORT, a flexible multi-readout transcription reporter vector for fission yeast that employs an enhanced GFP-lacZ fusion and a customizable minimal promoter. With pREPORT, gene expression driven by the transcription factor of interest can be quantified in a number of ways, both in live cells and in vitro, using a single reporter construct.
- MeSH
- beta-galaktosidasa analýza MeSH
- genetické vektory MeSH
- regulace genové exprese u hub * MeSH
- reportérové geny * MeSH
- Schizosaccharomyces pombe - proteiny genetika metabolismus MeSH
- Schizosaccharomyces genetika MeSH
- stanovení celkové genové exprese metody MeSH
- transkripční faktory genetika metabolismus MeSH
- zelené fluorescenční proteiny analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Every cell cycle iteration culminates with the resolution of a mitotic nucleus into a pair of daughter nuclei, which are distributed between the two daughter cells. In the fission yeast Schizosaccharomyces pombe, the faithful division of a mitotic nucleus depends on unperturbed lipogenesis. Upon genetically or chemically induced perturbation of lipid anabolism, S. pombe cells fail to separate the two daughter nuclei and subsequently initiate lethal cytokinesis resulting in the so-called "cut" terminal phenotype. Evidence supporting a critical role of lipid biogenesis in successful mitosis in S. pombe has been accumulating for almost two decades, but the exact mechanism explaining the reported observations had been elusive. Recently, several studies established a functional link between biosynthesis of structural phospholipids, nuclear membrane growth, and the fidelity of "closed" mitosis in S. pombe. These novel insights suggest a mechanistic explanation for the mitotic defects characteristic for some S. pombe mutants deficient in lipid anabolism and extend our knowledge of metabolic modulation within the context of the cell cycle. In this review, we cover the essential role of lipogenesis in "closed" mitosis, focusing mainly on S. pombe as a model system.
During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.
- MeSH
- centromera MeSH
- histonový kód MeSH
- homologní rekombinace * MeSH
- nukleozomy metabolismus MeSH
- proteiny buněčného cyklu antagonisté a inhibitory metabolismus MeSH
- regulace genové exprese u hub * MeSH
- represorové proteiny fyziologie MeSH
- Schizosaccharomyces pombe - proteiny antagonisté a inhibitory metabolismus fyziologie MeSH
- Schizosaccharomyces genetika MeSH
- transkripční faktory antagonisté a inhibitory metabolismus MeSH
- umlčování genů * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Epithelial-mesenchymal transition (EMT) is a cellular mechanism used by cancer cells to acquire migratory and stemness properties. In this study, we show, through in vitro, in vivo, and 3D culture experiments, that the mitochondrial protein LACTB manifests tumor suppressor properties in ovarian cancer. We show that LACTB is significantly down-regulated in epithelial ovarian cancer cells and clinical tissues. Re-expression of LACTB negatively effects the growth of cancer cells but not of non-tumorigenic cells. Mechanistically, we show that LACTB leads to differentiation of ovarian cancer cells and loss of their stemness properties, which is achieved through the inhibition of the EMT program and the LACTB-dependent down-regulation of Snail2/Slug transcription factor. This study uncovers a novel role of LACTB in ovarian cancer and proposes new ways of counteracting the oncogenic EMT program in this model system.
- MeSH
- beta-laktamasy * genetika metabolismus MeSH
- epitelo-mezenchymální tranzice * genetika MeSH
- karcinogeneze MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- mitochondriální proteiny genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádory vaječníků * genetika metabolismus patologie MeSH
- rodina transkripčních faktorů Snail * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast Saccharomyces cerevisiae, about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. S. cerevisiae features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in S. cerevisiae using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5' and 3' splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions.
BACKGROUND: Transcription factors of the CSL (CBF1/RBP-Jk/Suppressor of Hairless/LAG-1) family are key regulators of metazoan development and function as the effector components of the Notch receptor signalling pathway implicated in various cell fate decisions. CSL proteins recognize specifically the GTG[G/A]AA sequence motif and several mutants compromised in their ability to bind DNA have been reported. In our previous studies we have identified a number of novel putative CSL family members in fungi, organisms lacking the Notch pathway. It is not clear whether these represent genuine CSL family members. METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of in vitro and in vivo approaches we characterized the DNA binding properties of Cbf11 and Cbf12, the antagonistic CSL paralogs from the fission yeast, important for the proper coordination of cell cycle events and the regulation of cell adhesion. We have shown that a mutation of a conserved arginine residue abolishes DNA binding in both CSL paralogs, similar to the situation in mouse. We have also demonstrated the ability of Cbf11 and Cbf12 to activate gene expression in an autologous fission yeast reporter system. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the fission yeast CSL proteins are indeed genuine family members capable of functioning as transcription factors, and provide support for the ancient evolutionary origin of this important protein family.
- MeSH
- aktivní transport - buněčné jádro MeSH
- buněčný cyklus MeSH
- DNA fungální metabolismus MeSH
- konzervovaná sekvence MeSH
- mutace MeSH
- reportérové geny genetika MeSH
- responzivní elementy genetika MeSH
- Schizosaccharomyces pombe - proteiny chemie genetika metabolismus MeSH
- Schizosaccharomyces cytologie genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- sekvenční homologie aminokyselin MeSH
- transkripční faktory chemie genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Within a eukaryotic cell, both lipid homeostasis and faithful cell cycle progression are meticulously orchestrated. The fission yeast Schizosaccharomyces pombe provides a powerful platform to study the intricate regulatory mechanisms governing these fundamental processes. In S. pombe, the Cbf11 and Mga2 proteins are transcriptional activators of non-sterol lipid metabolism genes, with Cbf11 also known as a cell cycle regulator. Despite sharing a common set of target genes, little was known about their functional relationship. This study reveals that Cbf11 and Mga2 function together in the same regulatory pathway, critical for both lipid metabolism and mitotic fidelity. Deletion of either gene results in a similar array of defects, including slow growth, dysregulated lipid homeostasis, impaired cell cycle progression (cut phenotype), abnormal cell morphology, perturbed transcriptomic and proteomic profiles, and compromised response to the stressors camptothecin and thiabendazole. Remarkably, the double deletion mutant does not exhibit a more severe phenotype compared to the single mutants. In addition, ChIP-nexus analysis reveals that both Cbf11 and Mga2 bind to nearly identical positions within the promoter regions of target genes. Interestingly, Mga2 binding appears to be dependent on the presence of Cbf11 and Cbf11 likely acts as a tether to DNA, while Mga2 is needed to activate the target genes. In addition, the study explores the distribution of Cbf11 and Mga2 homologs across fungi. The presence of both Cbf11 and Mga2 homologs in Basidiomycota contrasts with Ascomycota, which mostly lack Cbf11 but retain Mga2. This suggests an evolutionary rewiring of the regulatory circuitry governing lipid metabolism and mitotic fidelity. In conclusion, this study offers compelling support for Cbf11 and Mga2 functioning jointly to regulate lipid metabolism and mitotic fidelity in fission yeast.
- MeSH
- metabolismus lipidů * genetika MeSH
- mitóza * genetika MeSH
- regulace genové exprese u hub * MeSH
- Schizosaccharomyces pombe - proteiny * genetika metabolismus MeSH
- Schizosaccharomyces * genetika metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Fission yeast 'cut' mutants show defects in temporal coordination of nuclear division with cytokinesis, resulting in aberrant mitosis and lethality. Among other causes, the 'cut' phenotype can be triggered by genetic or chemical perturbation of lipid metabolism, supposedly resulting in shortage of membrane phospholipids and insufficient nuclear envelope expansion during anaphase. Interestingly, penetrance of the 'cut' phenotype in mutants of the transcription factor cbf11 and acetyl-coenzyme A carboxylase cut6, both related to lipid metabolism, is highly dependent on growth media, although the specific nutrient(s) affecting 'cut' occurrence is not known. In this study, we set out to identify the growth media component(s) responsible for 'cut' phenotype suppression in Δcbf11 and cut6-621 cells. We show that mitotic defects occur rapidly in Δcbf11 cells upon shift from the minimal EMM medium ('cut' suppressing) to the complex YES medium ('cut' promoting). By growing cells in YES medium supplemented with individual EMM components, we identified ammonium chloride, an efficiently utilized nitrogen source, as a specific and potent suppressor of the 'cut' phenotype in both Δcbf11 and cut6-621. Furthermore, we found that ammonium chloride boosts lipid droplet formation in wild-type cells. Our findings suggest a possible involvement of nutrient-responsive signaling in 'cut' suppression.
- MeSH
- acetyl-CoA-karboxylasa genetika MeSH
- chlorid amonný chemie metabolismus farmakologie MeSH
- fenotyp MeSH
- kultivační média chemie MeSH
- lipidová tělíska účinky léků metabolismus MeSH
- metabolismus lipidů účinky léků genetika MeSH
- mitóza účinky léků genetika MeSH
- mutace MeSH
- penetrance MeSH
- Schizosaccharomyces pombe - proteiny genetika MeSH
- Schizosaccharomyces účinky léků genetika růst a vývoj metabolismus MeSH
- transkripční faktory genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Microbial colony growth can serve as a useful readout in assays for studying complex genetic interactions or the effects of chemical compounds. Although computational tools for acquiring quantitative measurements of microbial colonies have been developed, their utility can be compromised by inflexible input image requirements, non-trivial installation procedures, or complicated operation. Here, we present the Spotsizer software tool for automated colony size measurements in images of robotically arrayed microbial colonies. Spotsizer features a convenient graphical user interface (GUI), has both single-image and batch-processing capabilities, and works with multiple input image formats and different colony grid types. We demonstrate how Spotsizer can be used for high-throughput quantitative analysis of fission yeast growth. The user-friendly Spotsizer tool provides rapid, accurate, and robust quantitative analyses of microbial growth in a high-throughput format. Spotsizer is freely available at https://data.csiro.au/dap/landingpage?pid=csiro:15330 under a proprietary CSIRO license.
Ribosomal protein genes (RPGs) in Saccharomyces cerevisiae are a remarkable regulatory group that may serve as a model for understanding genetic redundancy in evolutionary adaptations. Most RPGs exist as pairs of highly conserved functional paralogs with divergent untranslated regions and introns. We examined the roles of introns in strains with various combinations of intron and gene deletions in RPL22, RPL2, RPL16, RPL37, RPL17, RPS0, and RPS18 paralog pairs. We found that introns inhibited the expression of their genes in the RPL22 pair, with the RPL22B intron conferring a much stronger effect. While the WT RPL22A/RPL22B mRNA ratio was 93/7, the rpl22aΔi/RPL22B and RPL22A/rpl22bΔi ratios were >99/<1 and 60/40, respectively. The intron in RPL2A stimulated the expression of its own gene, but the removal of the other introns had little effect on expression of the corresponding gene pair. Rpl22 protein abundances corresponded to changes in mRNAs. Using splicing reporters containing endogenous intron sequences, we demonstrated that these effects were due to the inhibition of splicing by Rpl22 proteins but not by their RNA-binding mutant versions. Indeed, only WT Rpl22A/Rpl22B proteins (but not the mutants) interacted in a yeast three-hybrid system with an RPL22B intronic region between bp 165 and 236. Transcriptome analysis showed that both the total level of Rpl22 and the A/B ratio were important for maintaining the WT phenotype. The data presented here support the contention that the Rpl22B protein has a paralog-specific role. The RPL22 singleton of Kluyveromyces lactis, which did not undergo whole genome duplication, also responded to Rpl22-mediated inhibition in K. lactis cells. Vice versa, the overproduction of the K. lactis protein reduced the expression of RPL22A/B in S. cerevisiae. The extraribosomal function of of the K. lactis Rpl22 suggests that the loop regulating RPL22 paralogs of S. cerevisiae evolved from autoregulation.
