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The relationship between splicing and posttranslational modifications of chromatin in Saccharomyces cerevisiae
Kovaľová, Libuša ; Folk, Petr (advisor) ; Čáp, Michal (referee)
Protein Prp45, the yeast ortholog of the human transcription coregulator SNW1/SKIP, has been previously associated only with the regulation of pre-mRNA splicing. However, our laboratory found that protein Prp45 genetically interacts not only with the proteins involved in pre-mRNA splicing, but also with factors important for transcription elongation and with chromatin modifying enzymes. Our data and the information about the human ortholog SNW1/SKIP suggest that Prp45 could serve as a regulator coupling splicing, transcription and chromatin state in S. cerevisiae. The main aim of this diploma thesis was to find out whether the protein Prp45, which is essential for cotranscriptional assembly of the spliceosome, affects posttranslational modifications of chromatin on transcribed genes. Using chromatin immunoprecipitation, the influence of prp45(1-169) mutation on trimethylation of histone H3 at lysine 4 and acetylation of histone H3 at lysines 9, 14 and 18 on transcriptionally active genes was not confirmed. The other aim was to analyse the behavior of cells synchronized by α-factor by using flow cytometry. According to our results, prp45(1-169) mutation leads to the prolongation of the cell cycle. For the purpose of monitoring the dynamics of nucleosomes in S. cerevisiae strains, the system of...
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CK1 family kinases and their role in Wnt signalling
Horázná, Daniela ; Macůrková, Marie (advisor) ; Folk, Petr (referee)
Wnt signalizace je velmi důležitou drahou pro vývoj a růst buněk. V člověka obsahuje šest proteinů různých funkcí a struktur, kódovaných různými geny. Signalizační kaskáda začíná vazbou molekuly Wnt na receptory cílové buňky. Tím je zahájen složitý řetězec navazujících a vzájemně se ovlivňujících reakcí, poháněný a zároveň inhibovaný aktivitou CK1 kináz. Každá kináza má v ignalizaci jinou funkci, ale jejich funkce se mohou překrývat. Za určitých podmínek mohou některé změnit své chování z aktivačního na inhibiční. Tato práce se pokouší rozebrat CK1 členy jednotlivě a ukazuje jejich úlohy ve Wnt signalizaci podle nejnovějších vědeckých poznatků.
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Transcriptional regulation of GAL genes - model for chromatin structure study
Brožková, Anita ; Folk, Petr (advisor) ; Konířová, Jana (referee)
The GAL system in Saccharomyces cerevisiae enables this budding yeast to metabolize galactose. Expression of GAL genes is controlled by a regulatory cascade in which galactose triggers the activation of GAL gene expression, whereas glucose acts as a repressor. GAL genes in yeast have been used for decades as a model system for transcription regulation in eucaryotes. The products of GAL genes are GAL regulatory and GAL structural genes. Intriguingly, during studies of the GAL system it has been discovered that one of the regulatory genes and the structural gene for the galactokinase enzyme are apparently related. It has been suggested that an ancestor of the two genes underwent a gene duplication event which allowed the paralogs to gain different functions. The GAL genes serve as a model system for the study of chromatin changes during transcription activation or repression. Transcriptional repression of GAL1 and GAL10 genes via ncRNA represents one of the recently discovered regulatory mechanisms of the GAL system. This mechanism has been discovered due to the changes in the histone methylation pattern across the GAL10 locus. However, the latest discovery in GAL gene regulation has probably been the role of nuclear localization of the GAL1,-7,-10 cluster in response to changes of carbon sources. GAL genes...
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Chromatin modifiers and their relation to transcription regulation in Saccharomyces cerevisiae
Hálová, Martina ; Folk, Petr (advisor) ; Staněk, David (referee)
Relations among transcription, pre-mRNA processing and chromatin modifications are only partially understood. The human protein SNW1/SKIP belongs to factors which couple these processes. The protein plays role in pre-mRNA splicing and transcription on the level of both initiation and elongation. According to the hypothesis of K. Jones laboratory, it physically and functionally interacts with positive transcription elongation factor b during transcription elongation and influences methylation of histone H3 on lysine 4, a modification characteristic for active transcription (Bres et al., Genes Dev. 19:1211-26, 2005, Bres et al., Mol Cell. 36:75-87, 2009). The yeast ortholog of SNW1/SKIP, Prp45, was until now reported only in connection with splicing regulation. However, unpublished results from our Laboratory and others showed that it is employed in transcription elongation as well. The aim of the diploma project was to search for the relations between Prp45 and the factors regulating transcription. It was confirmed that the mutation prp45(1 169) results in the delay of PHO5 and PHO84 expression during transcriptional induction. Next, we discovered new genetic interactions between PRP45 and several genes encoding the effectors of chromatin modifications. How Prp45 influences the expression of PHO5 and PHO84...
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Vlastnosti DNA vazebných mutant proteinů CSL
Teska, Mikoláš ; Folk, Petr (advisor) ; Šťovíček, Vratislav (referee)
Notch pathway plays a critical role during the development and life of metazoan organisms. CSL proteins are the component of the Notch pathway that mediates the regulation of target genes. The discovery of CSL-like proteins in yeast raised the question of their function in unicellular organisms which did not utilize the canonical Notch pathway. CSL-homologues in yeast are conserved in parts that are important for DNA binding and for fission yeast proteins it was shown that they bind to CSL recognition elements in vitro. In fission yeast, CSL paralogues Cbf11 and Cbf12 play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Yeast CSL proteins have long and intrinsically unstructured N- terminal domains compared to metazoan CSL proteins. In this study, we investigated the functional significance of these extended N-termini of CSL proteins by their complete removal. For newly constructed truncated variants of proteins Cbf11 and Cbf12 in Schizosaccharomyces pombe we observed the lack of ability to bind CSL recognition RBP probe. The removal of N-terminal parts of CSL proteins in fission yeast led to the change in their cellular localization. Once strongly preferred nuclear localization changed by the removal of N-terminal domains to cytoplasmic localization with a...
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Notch-independent functions of CSL transcription factors
Teska, Mikoláš ; Folk, Petr (advisor) ; Převorovský, Martin (referee) ; Kuthan, Martin (referee)
Notch pathway plays a critical role during development and life of Metazoan organisms. CBF1 is a component of the Notch pathway that mediates the regulation of target genes. The discovery of CBF1-like proteins in yeast raised the question of their function in unicellular organisms - before the origin of canonical Notch pathway. CBF1-homologs in yeast are conserved in parts that are important for DNA binding and bind to CBF1-binding elements in vitro. CBF1 and related transcription factors in Metazoa (CSL) interact with many proteins in Notch-dependent as well as Notch-independent complexes. The Notch receptor has likewise some CSL-independent functions. This assay reports about interacting partners of CSL in Metazoa along with homologous proteins in yeast with the aim to highlight potential interactions of CBF1-homologs in evolutionary ancestral context.
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Mechanisms of invasiveness and transcription regulation in cancer cells
Tolde, Ondřej ; Folk, Petr (advisor) ; Kovář, Jan (referee) ; Brdička, Tomáš (referee)
The mechanisms of invazivity and regulation of transcription of cancer cells Cancer originates in cells that overcome the control mechanisms of the organism. Cancer cells can be eventually released from the site of origin and spread through tissues. Cancer cells can acquire certain mechanisms that enable them to more effectively invade surrounding tissue or layers of other cells. The research on the migration of cancer cells is important for the understanding of the origin and spreading of metastases and consequently for anticancer therapy. In my Ph.D. work, I participated in the research of the properties of invasive metastatic cells. We compared non-invasive rat sarcoma cell line with a higly metastatic cell line derived from it. We showed that cells of the invasive cell line use amoeboid mode of migration, have upregulated Rho/ROCK signaling, and have accumulated actin and myosin at the leading edge. It is at the leading edge where the cells generate their traction forces. Cells of non-invasive cell line use mesenchymal mode of migration and generate forces mainly at their retracting end. We also compared two breast cancer cell lines derived from a single carcinoma. We showed that the more invasive cell line, derived from its parental line by neoplastic transformation, displayed elevated cytoskeletal...
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New aspects of the cell submembrane signaling
Heneberg, Petr ; Dráber, Petr (advisor) ; Bilej, Martin (referee) ; Folk, Petr (referee)
This dissertation contributes to elucidation of some mechanisms of the mammalian cell submembrane signaling. Major part of the research was conducted on mast cells and basophils activated via the high affinity IgE receptor, FcεRI, or via the cell surface glycoprotein Thy-1. New roles of actin cytoskeleton in mast cell signaling via FcεRI and Thy-1 are described. Discovery of new transmembrane adaptor protein non-T cell activation linker, NTAL, short time before the initiation of work on the thesis led to the increased attention paid to this protein. Dramatic changes of signaling in mast cells deficient in NTAL, or with up- or down-regulated expression of this protein are described. NTAL was also found to be one of proteins phosphorylated following the Thy-1 aggregation. Spatiotemporal distribution of surface glycoprotein Thy-1 at different levels of resolution and some biochemical properties of cells activated via Thy-1 are depicted. Screen for nonreceptor hitherto unknown protein tyrosine phosphatases in mast cells and basophils was conducted and initial analysis of spatiotemporal distribution and function of phosphatase PTP20 in mast cell signaling was performed. Next, the role of reactive oxygen and nitrogen species in the regulation of mast cell protein tyrosine phosphatases was summarized. New...
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Physical interactions of the splicing factor Prp45
Kratochvílová, Eliška ; Folk, Petr (advisor) ; Doubravská, Lenka (referee)
It is well known that chromatin posttranslational state, transcription and splicing influence each other. Nevertheless, the details of this coupling are not fully understood. In S. cerevisiae, it is possible to induce conditions, in which splicing is uncoupled from transcription. Such situation occurred in cells expressing a mutated splicing factor Prp45, whose human homolog has been proved to participate in transcription regulation and also in splicing reactions. Based on previously indicated interactions in high throughput two-hybrid screens, we have been looking for physical links between Prp45 and proteins involved in chromatin posttranslational modifications. Finding of such a link would provide insight into the relationships of gene expression processes. Using coimmunoprecipitation and affinity purification, we were unable to detect physical interactions between Prp45 and our candidate chromatin regulators. Alternative approaches are discussed. Using the precipitation techniques, we mapped the interaction of Prp46 with truncated variants of Prp45. This observation contributes to our knowledge of protein-protein interactions within the spliceosome.
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