Chez Schizosaccharomyces pombe, le cycle méiotique est le mode de division cellulaire spécialisé qui permet la formation d’ascospores résistantes à différents stress lorsque les conditions environnementales ne sont pas propices à la multiplication cellulaire. Lors de mes travaux de thèse, mes objectifs consistaient à caractériser le rôle et le mécanisme d’action de la protéine Cuf2 lors du cycle méiotique chez S. pombe. Mes résultats ont montré que le gène cuf2[indice supérieur +] était exprimé exclusivement lors des divisions méiotiques et que la protéine se co-localisait de manière constitutive avec le matériel génétique. De plus, mes résultats ont dévoilé que Cuf2 participait à l’activation et à la répression de plusieurs gènes méiotiques selon un mécanisme de nature transcriptionnelle en s’associant spécifiquement avec leur région promotrice. Par la suite, mes résultats ont mis en évidence que Cuf2 interagissait physiquement avec Mei4, un facteur de transcription méiose-spécifique, au noyau des cellules méiotiques. Notamment, mes résultats ont montré que la présence de Mei4 et de son motif de liaison à l’ADN dénommé FLEX étaient nécessaires afin que Cuf2 puisse s’associer au promoteur de son gène cible fzr1[indice supérieur +] afin d’en activer l’expression. L’ensemble de mes résultats indiquent que Cuf2 et Mei4 interagissent aux promoteurs de certains gènes lors des divisions méiotiques afin d’en co-activer l’expression. D’ailleurs, mes résultats ont également montré que la fonction de Cuf2 était importante à la formation d’ascospores et à leur viabilité ; en absence de Cuf2, la majorité des ascospores présentent diverses aberrations et plus de la moitié d’entre elles sont non-viables. Globalement, mes résultats démontrent que Cuf2 est un régulateur critique de l’expression génique lors du cycle méiotique et que cette fonction est essentielle à la sporulation chez S. pombe.

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Thesis advisor: Clare O'Connor / Within cells, reactive oxygen species (ROS) are synthesized naturally and in response to environmental stimuli. However, ROS have deleterious effects on a wide range of cellular molecules. Oxidative stress, caused by the ROS generated by the partial reduction of oxygen, is a major cause of cell damage linked to the initiation and progression of numerous diseases. Thioredoxin peroxidase (Tpx1) plays important roles in cellular defense against ROS. Although homologous genes and their functions have been identified in other eukaryotes, the level of activity as well as the necessity of this protective enzyme in S. pombe exposed to oxidative stress has yet to be fully elucidated. To explore the role of the Tpx1 protein in oxidative stress resistance, novel strains were constructed in which the tpx1 gene was overexpressed. The polymerase chain reaction was used to amplify txp1, and the amplified sequence was cloned into the yeast overexpression plasmid, pNMT41, which allows overexpression under the control of the powerful promoter. DNA sequencing was used to determine that the sequences had been properly inserted into the vector. The plasmids were transformed into two leu- yeast strains: FWP6 and TP108-3C. Production of the Tpx1 protein was ensured using Western Blot techniques. Experimentation to test the responses of the tpx1 strain to oxidative stress will employ a variety of reactive oxygen generators, including hydrogen n peroxide, menadione, tert-butyl hydroperoxide, and paraquat. The results generally supported the proposed role of Tpx1 to confer additional resistance against the oxidative stress. In a complementary line of investigation, knockout strains are being constructed to reduce the levels of the Tpx1 in S. pombe. Gene deletion cassettes were constructed for tpx1. Currently, the strains are being analyzed for the successful replacement of the endogenous tpx1 gene by homologous recombination. If the absence of the protein results in decreased cell viability, the role of Tpx1 indicated by the overexpression experiments could be supported. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Thesis advisor: Charles S. Hoffman / Transcription of the Schizosaccharomyces pombe fbp1 gene is subject to repression by environmental glucose that is detected through a cAMP signaling pathway. Previous research has identified several glucose insensitive transcription (git) genes that are required for glucose repression. One of these genes, git7, is required for cAMP signaling, but its exact function in the process is unknown. Analysis of this gene is complicated because it carries out additional essential functions that are unrelated to cAMP signaling. The git7-93 allele encodes a protein that possesses an18 amino acid duplication in the carboxy-terminus and only displays a defect in cAMP signaling, while two other mutant alleles that affect all Git7-dependent processes alter residues in the amino-terminus. My thesis work was composed of two projects designed to investigate the role of the Git7 carboxy-terminus in cAMP signaling and to identify more precisely the carboxy-terminal domain that is required for cAMP signaling. The first part of my study involved a genetic screen to isolate and characterize mutations that suppress the git7-93 allele, but fail to suppress a git7 mutant allele in which the amino-terminus of the protein is altered. Such allele-specific suppression may identify the direct target of Git7 function in the cAMP pathway. I have isolated a collection of 55 suppressor strains. Molecular and genetic analyses of these strains have reduced this number to nine candidates, which could have represented allele specific suppressors. However, the final genetic screen determined that all the candidates were extragenic suppressors. The second part of my study involved a screen for new mutant alleles of git7 that resemble git7-93 in that they confer a cAMP signaling defect, but do not confer cell wall or septation defects caused by the git7-27 or git7-235 alleles. This was carried out by performing a random mutagenesis on a cloned wild type git7 gene and then screening for plasmids that could suppress the cell wall or septation defects, but not the cAMP signaling defect, in a git7-235 host strain. Taken together, these two studies should help to identify the role that git7 plays in the cAMP signaling pathway Schizosaccharomyces pombe. / Thesis (BS) — Boston College, 2004. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

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Thèse (M.Sc.)--Université Laval, 2005. / Titre de l'écran-titre (visionné le 23 février 2006). Bibliogr.

<p>The capability to control the progression of the cell cycle, including the means to enter into a stable non-proliferative state, is essential for eukaryotic unicellular and multicellular organisms. A quiescent state similar to G0 of higher eukaryotes can be induced by nitrogen starvation of the fission yeast model organism Schizosaccharomyces pombe. Using high-resolution tiling arrays for genome-wide transcriptional profiling we explore the early transcriptional reprogramming on the route to quiescence. Furthermore, we demonstrate that cells carrying a mutation in the high mobility group (HMG) box of the histone lysine demethylase spLsd1 fail to acquire characteristics of quiescent cells and rapidly lose viability under nitrogen-starved conditions. Since no such defect is seen as a result of catalytic inactivation, the HMG domain of spLsd1 seems to confer a function to the protein that is independent of the histone demethylase activity. We show that the HMG domain of spLsd1 is required for transcriptional activation and repression of a large set of genes, both during vegetative growth and on the route to quiescence. We also confirm that spLsd1 is a repressor of antisense transcription, and that this function is at least partially dependent on the HMG domain of the protein.</p>

The capability to control the progression of the cell cycle, including the means to enter into a stable non-proliferative state, is essential for eukaryotic unicellular and multicellular organisms. A quiescent state similar to G0 of higher eukaryotes can be induced by nitrogen starvation of the fission yeast model organism Schizosaccharomyces pombe. Using high-resolution tiling arrays for genome-wide transcriptional profiling we explore the early transcriptional reprogramming on the route to quiescence. Furthermore, we demonstrate that cells carrying a mutation in the high mobility group (HMG) box of the histone lysine demethylase spLsd1 fail to acquire characteristics of quiescent cells and rapidly lose viability under nitrogen-starved conditions. Since no such defect is seen as a result of catalytic inactivation, the HMG domain of spLsd1 seems to confer a function to the protein that is independent of the histone demethylase activity. We show that the HMG domain of spLsd1 is required for transcriptional activation and repression of a large set of genes, both during vegetative growth and on the route to quiescence. We also confirm that spLsd1 is a repressor of antisense transcription, and that this function is at least partially dependent on the HMG domain of the protein.

Inorganic phosphate is an essential nutrient required by all organisms for optimal growth. During phosphate starvation, Saccharomyces cerevisiae induces a set of genes responsible for the regulation of inorganic phosphate acquisition. The phosphate-responsive signaling (PHO) pathway controls this response, with the CDK-cyclin complex Pho85-Pho80 playing a prominent role. Here we report the X-ray structure of the Pho85-Pho80 complex, identifying the unique structural features that distinguish it from other cell cycle associated CDK-cyclin complexes. The structure reveals a specific salt bridge between a Pho85 arginine and a Pho80 aspartate that maintains a Pho80 loop confirmation important for substrate recognition and makes phosphorylation of the Pho85 activation loop dispensible. We show that a cluster of residues distal to the kinase active site are involved in a high affinity interaction between the Pho80 cyclin and the transcription factor substrate (Pho4). The structure also reveals a separate high affinity binding site for the CDK inhibitor (Pho81). The fission yeast, Schizosaccharomyces pombe, regulates expression of the secreted acid phosphatase \((pho1^+)\) via a non-orthologous PHO pathway. The genes induced by phosphate limitation and the molecular mechanism by which the genetically identified positive \((pho7^+)\) and negative \((csk1^+)\) regulators function are not known. Here we use a combination of molecular biology, expression microarrays, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq), and global transcriptome sequencing (RNA-Seq) to characterize the role of \(pho7^+\) and \(csk1^+\) in the PHO response. We show that there is a fast and slow response to phosphate starvation, each with defined regulatory roles. We use ChIP-Seq to identify members of the Pho7 regulon and characterize Pho7 binding dynamics in response to phosphate-limitation and Csk1 activity. We identify a conserved PHO response for the PHO5 \((pho1^+)\), PHO84 \((spbc8e4.01c^+)\), and GIT1 \((spbc1271.09^+)\) orthologs. We show that activation of \(pho1^+\) requires Pho7 binding to a UAS in the \(pho1^+\) promoter and that a URS is necessary for Csk1 repression. We find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require \(pho7^+\) for maximal induction. Using RNA-Seq we show that Pho7 is also involved in regulating non-coding transcription and is a bi-functional transcription factor.

Spt6 is a conserved eukaryotic transcription factor, known to interact with both nucleosomes and RNA polymerase II (RNAPII) to control transcription. We have initiated study of Spt6 in S. pombe in order to identify both novel and conserved roles in regulation of transcription and chromatin. We first constructed and analyzed spt6 mutants by several approaches. As Spt6 is known to be required for histone H3K36 methylation in both Saccharomyces cerevisiae and human cells, we examined the global levels of several histone modifications; we found that in S. pombe, Spt6 is required for both H3K4 and H3K36 trimethylation. We examined the chromatin state at two highly expressed genes, \(act1^+\) and \(pma1^+\), and found that there is a defect in recruitment of the methyltransferases responsible for those marks, Set1 and Set2, respectively. We also observed loss of nucleosomes, as well as a decrease in histone H2B monoubiquitylation. These results suggest that Spt6 plays an important role in chromatin regulation during transcription. We also conducted transcriptional analysis of an spt6 mutant by both microarray and high-throughput sequencing (RNA-seq) and discovered that Spt6 plays a critical role in maintaining the integrity of transcription genome-wide. We found that Spt6 is required to repress antisense transcription, with nearly 70% of genes having antisense transcripts increased by at least two-fold in an spt6 mutant. We also found that transcription of most long terminal repeats (LTRs) is derepressed. Finally, we found that a major class of transcripts elevated in the spt6 mutant is derived from heterochromatin, which is normally silenced. To study the heterochromatic silencing defect in greater detail, we analyzed the chromatin state of the pericentric repeats and found a decrease in H3K9 trimethylation, elevated levels of H3K14 acetylation, reduced recruitment of several known silencing factors and a loss of siRNA production. We also see a very modest increase in RNAPII recruitment. Based on this combination of phenotypes, Spt6 is likely to contribute to both transcriptional and post-transcriptional silencing mechanisms. Taken together, we have found that Spt6 plays several important roles to control transcription in both euchromatin and heterochromatin in S. pombe.