Dissertations / Theses on the topic 'Saccharomyces cerevisiae, cell cycle, Snf1'

The AMP-activated protein kinase (AMPK) family is a group of Serine/Threonine kinases highly conserved in eukaryotes, from yeast and insects to plants and mammals. Their primary role is the integration of signals regarding nutrient availability and environmental stresses, ensuring the adaptation to those conditions and cell survival (Hardie G., 2007; Ghillebert R. et al., 2011). As its homologue AMPK, in Saccharomyces cerevisiae Snf1 exists as a heterotrimeric complex. Core of this enzyme is the catalytic α subunit (Snf1), made up of a canonical catalytic domain in its N-terminus and of an autoinhibitory C-terminal domain which mediates the interaction with the regulatory subunits of this kinase (Rudolph M.J. et al., 2005). These subunits are: the β subunit (Sip1, Sip2 and Gal83, alternatively), which regulates Snf1 localization (Vincent O. et al., 2000) and the γ subunit (Snf4) that, interacting with the autoinhibitory domain of Snf1, guarantees the complete activation of the kinase (Momcilovic M. et al., 2008). Beyond the interaction with Snf4, the activation of the protein kinase Snf1 is determined by the phosphorylation of Thr210 residue in the α subunit (McCartney R.R. and Schmidt M.C., 2001). Three upstream kinases (Sak1, Tos3, Elm1) are responsible for such a phosphorylation. Those kinases are constitutively active, but metabolic signals, such as high glucose concentrations, promote the activity of the phosphatase complex Reg1/Glc7 which dephosphorylates and hence inactivates Snf1 (Huang D. et al.,1996; Sanz P. et al., 2000; Sutherland C. et al., 2003; Hong S. et al., 2003). In budding yeast, Snf1 is required for adaptation to glucose limitation and for growth on non-fermentable carbon sources. In those conditions Snf1 controls the expression of more than 400 genes. Apart from carbon metabolism, Snf1 affects several other processes; in fact, this kinase controls the expression of some important genes involved in the resistance to different environmental stresses (osmotic and alkaline stresses) or in the regulation of different cellular processes such as sporulation, aging, filamentous and invasive growth (Portillo F. et al., 2005; Ashrafi K. et al., 2000; Vyas V.K. et al., 2003). As a transcriptional regulator, Snf1 exerts its role modulating gene transcription at different levels. This kinase regulates different transcription factors, such as the transcription inhibitor Mig1 (Treitel M.A. et al., 1998; Papamichos-Choronaris M. et al., 2004) or some other transcription factors like Adr1, Sit4, Cat8 and Gcn4 which regulate the expression of genes involved in central metabolic functions, such as gluconeogenesis and respiration (Hedbacker K. and Carlson M., 2008; Smets B. et al., 2010; Kacherovsky N. et al., 2008). Moreover, protein kinase Snf1 is even able to positively regulate the transcription of some metabolic genes influencing the chromatin remodelling process and the recruitment of some Pre-Initiation Complex (PIC) components at promoters. In fact, Snf1 promotes acetylation of histone H3 by either the direct phosphorylation of Ser10 of histone H3 and the phosphorylation of acetyl-tranferase Gcn5 (Lo W. et al., 2005; van Oevelen C.J. et al., 2006; Liu Y. et al., 2010). Moreover, Snf1 is involved in the recruitment to some promoters of Mediator complex (Young E.T. et al., 2002), SAGA complex (van Oevelen C.J. et al., 2006), TATA-binding protein (TBP) (Shirra M.K. et al., 2005) and RNA Pol II (Tachibana C. et al., 2007; Young R.T. et al., 2012). My PhD research activity was focused on the role of Snf1 in the regulation of the expression of G1-specific genes, and thus in its function as modulator of cell cycle progression. Data obtained in our laboratory showed that, in cells grown in 2% glucose, deletion of SNF1 gene caused a delayed G1/S phase transition, consistently with a decreased expression of CLB5 gene. In keeping with that defective expression, the snf1Δ strain showed a severe reduction of Clb5 protein levels and a consequent decrease of phosphorylation of Clb5/Cdk1 complex targets, such as Sld2, which are responsible for the onset of DNA replication. Moreover, our co-immunoprecipitation assays highlighted that Snf1 interacts with Swi6, the common subunit of SBF (Swi4-Swi6) and MBF (Mbp1-Swi6) transcription complexes which regulate the expression of G1-specific genes (Nasmyth, K. and Dirick, L., 1991; Koch C. et al., 1993). Remarkably, the phenotype of the snf1 null mutant was complemented by a glucose concentration higher than 2% (5%), suggesting that the role of Snf1 in the modulation of cell cycle progression could depend on the nutritional status of cells. Those data, published in Pessina S. et al., 2010, newly indicated that Snf1 was involved in the regulation of G1/S transition and pointed to a role for this kinase in the modulation of G1-specific gene expression. To gain further insight into the function of Snf1, we then analyzed the expression profile of G1-specific genes in cells synchronized in G1 phase by α-factor treatment and released into fresh medium. Our analyses showed that loss of Snf1 (snf1Δ strain) severely affected the expression of CLN2, PCL1 (SBF-dependent) and CLB5, RNR1 (SBF-dependent) genes, suggesting that Snf1 regulates the expression of both SBF- and MBF-dependent genes. Although protein Snf1 was not detectable at promoters of G1-specific genes, we investigated whether it could modulate the activity of SBF and MBF complexes and we found that in a snf1Δ strain the recruitment of Swi6 to G1-specific promoters was affected. Moreover, our Chromatin ImmunoPrecipitation (ChIP) assays also showed that in a snf1Δ strain the defective association of Swi6 to promoters led to a decreased recruitment of both the FACT complex, which is involved in the chromatin remodelling at G1-specific promoters, and of the RNA Pol II. Since it is known that the subcellular localization of Swi6 influences its interaction with promoters, then we analyzed its localization in G1 synchronized cells. In keeping with literature data (Sidorova J.M. et al., 1995; Taberner, F.J. and Igual, J.C., 2010), our analyses showed that in wild type cells synchronized in G1 phase by α-factor treatment Swi6 was essentially nuclear. Instead, in a snf1Δ strain Swi6 was localized in the nucleus only in the 60% of the G1-arrested cells, consistently with the reduced binding of Swi6 to G1-specific promoters. It is well known that the Swi6 interaction to DNA is mediated by the DNA binding-proteins Swi4 and Mbp1 (Andrews B.J. and Moore L.A., 1992; Moll T. et al., 1992). Therefore we extended our analyses to those proteins and we found that also the nuclear localization and the subsequent binding to DNA of Swi4 and Mbp1 were affected in a snf1Δ strain. Therefore, our data provide a representative snapshot of what occurs in vivo in a snf1 null mutant, supporting the notion that Snf1 promotes the expression of G1-specific genes modulating the nuclear localization of SBF and MBF components and thus promoting the formation of a complete Pre-initiation Complex (PIC) at G1-specific promoters. It is well known that phosphorylation of Snf1 at Thr210 leads to the full activation of the kinase (Hong S.P. et al., 2003; Sutherland C.M. et al., 2003). Then, in order to obtain insight into the Snf1 molecular mechanism in cell cycle regulation, we investigated its phosphorylation on Thr210 during cell cycle progression. Snf1 was slightly phosphorylated on the Thr210 residue during all the cell cycle, suggesting that this kinase was partially active. To determine whether the activation of Snf1 was involved in its function as regulator of G1 transcription, we analyzed the expression of SBF- and MBF-dependent genes in the SNF1-T210A mutant and we found that in this mutant the expression of those genes was reduced. In keeping with those data, the expression of G1-specific genes resulted affected also in a SNF1-K84R mutant, in which the ATP binding site has been destroyed causing a severe reduction of Snf1 kinase activity. On the base of those findings, we investigated whether Snf1 could exert its role in G1 phase through the phosphorylation of specific substrates and we found that Snf1 phosphorylates in vitro Swi6 on Ser760. Nevertheless, analyses of site-specific mutants (SWI6-S760A or SWI6-S760E) did not show any alteration of G1/S transition, suggesting that this phosphorylation was not involved in the role of Snf1 as regulator of cell cycle. The ChIP analyses of Swi6 binding to CLN2 and RNR1 promoters, then, showed that in the SNF1-K84R mutant the recruitment of Swi6 was slightly affected; nevertheless, that alteration was not severe as that of a snf1Δ strain. Consistently, neither the recruitment of FACT complex nor the binding of RNA Pol II to G1-specific promoters was affected in the SNF1-K84R mutant. Since this last finding seemed to disagree with the severe reduction of mRNA expression of SBF- and MBF-dependent genes observed in the SNF1-K84R mutant, we wondered whether defects in transcriptional elongation might occur in that strain. Thus, we analyzed the occupancy of FACT complex and of RNA Pol II at the internal regions of CLN2 and RNR1 genes by ChIP analyses; in the SNF1-K84R mutant the occupancy of both those complexes was decreased, suggesting that the kinase activity of Snf1 promotes the transcriptional elongation across G1-specific genes. In conclusion, the sum of data here presented indicates that protein kinase Snf1 is involved at different levels in the modulation of the G1-specific gene expression, thus highlighting a new function for Snf1 in the regulation of G1/S transition.

Snf1 è una serina/treonina chinasi necessaria per il lievito S. cerevisiae per la crescita in condizioni di limitazione di nutrienti e per l’utilizzo di fonti di carbonio alternative al glucosio. Nel nostro laboratorio è stato precedentemente dimostrato che la mancanza di Snf1 causa un difetto nella transizione G1/S del ciclo cellulare e un difetto nell’espressione dei geni di fase G1 anche in condizioni di sufficienza nutrizionale (2% glucosio). È stato quindi approfondito il coinvolgimento di Snf1 in tre importanti processi cellulari: ciclo, trasduzione del segnale e metabolismo. Per dimostrare la necessità dell’attività catalitica di Snf1 per una corretta transizione G1/S è stato utilizzato un ceppo Snf1-I132G, in cui l’attività della chinasi può essere inibita utilizzando l’inibitore specifico 2NM-PP1. Il difetto nell’effettuare la transizione G1/S e nella trascrizione dei geni di fase G1 di questo ceppo in presenza dell’inibitore è stata dimostrata sia con esperimenti di rilascio da α-factor sia mediante elutriazione. Nello studio del coinvolgimento di Snf1 nella regolazione di altri pathway di trasduzione del segnale è stata identificata, mediante esperimenti di CoIP/MS, l’interazione tra Snf1 e l’adenilato ciclasi (Cyr1), l’enzima responsabile della produzione di AMP ciclico (cAMP), attivatore di PKA. Il dominio della proteina Cyr1 contenente il RAS Associating Domain e 2 putativi siti consenso di Snf1 è stato purificato in E.coli e ne è stata dimostrata la fosforilazione in vitro da parte della chinasi. È stato inoltre da dimostrato che in un ceppo Snf1-G53R, in cui la chinasi è costitutivamente attivata, si ha una riduzione di circa il 50% nel contenuto di cAMP intracellulare, assieme alla deregolazione dell’espressione di geni PKA-dipendenti. É stata quindi ipotizzata l’esistenza di un crosstalk fra i pathway di Snf1 e PKA. Per chiarire il ruolo globale di Snf1 in condizioni di sufficienza nutrizionale è stata effettuata un’analisi trascrittomica (gene-chip) di cellule wt e snf1∆ cresciute in 2% e 5% glucosio. É stato così evidenziato che la mancanza di Snf1 in 2% glucosio, ma non in 5%, causa la deregolazione di circa 1000 geni, fra i quali ad esempio i geni glicolitici. Sono pertanto state indagate le deregolazioni metaboliche presenti in cellule prive di Snf1. L’analisi dei metaboliti secreti da cellule snf1Δ in 2% glucosio ha permesso di dimostrare che, in relazione alla propria velocità di crescita, producono più etanolo ed acetato in confronto a cellule wt. Questa attività glicolitica maggiore del wt è abolita, coerentemente a quanto già osservato, in presenza di 5% glucosio. É stato quindi dimostrato che anche nelle condizioni di crescita dei nostri esperimenti cellule snf1Δ presentano un accumulo di acidi grassi, fenotipo già osservato con bassi livelli di glucosio e dovuto all’assenza di fosforilazione Snf1-dipendente dell’enzima acetil-CoA carbossilasi. Una più estesa analisi metabolica, sia mediante spettrometria di massa che mediante NMR, ha permesso di descrivere in dettaglio i riarrangiamenti metabolici che cellule snf1Δ subiscono perché la crescita sia garantita nonostante i processi anabolici sopra descritti. Cellule snf1Δ in 2% glucosio accumulano glutammato in funzione di un maggiore consumo degli amminoacidi forniti nel terreno, evento necessario per il mantenimento della velocità di crescita del mutante. Il mutante inoltre accumula intermedi del ciclo degli acidi tricarbossilici e se trattato con antimicina A, un inibitore del della catena di trasporto degli elettroni, subisce un effetto deleterio in 2%, ma non in 5% glucosio. Il trattamento influenza negativamente crescita e contenuto di ATP e causa nel mutante aumento di NADH, dimostrandone la mancata riossidazione mitocondriale.<br>Snf1 is a serine/threonine kinase required by the yeast S. cerevisiae to grow in nutrient-limited conditions and to utilize carbon sources alternative to glucose. In our laboratory we previously demonstrated that lack of Snf1 causes an impairment of the G1/S transition of the cell cycle and a defect in the expression of genes of the G1 phase, even in condition of glucose sufficiency (2% glucose). It was therefore investigated the involvement of Snf1 in three important cellular processes: cycle, signal transduction and metabolism. To demonstrate the necessity of the catalytic activity of Snf1 for a proper G1/S transition was utilized a Snf1-I132G strain, in which the catalytic activity of the kinase can be inhibited by the specific inhibitor 2NM-PP1. The impairment of the G1/S transition and of the transcription of G1 genes in this strain in the presence of the inhibitor was demonstrated performing α-factor release and elutriation experiments. Studying the involvement of Snf1 in the regulation of other signaling pathways it was identified, through CoIP/MS experiments, the interaction between Snf1 and adenylate cyclase (Cyr1), the enzyme responsible for the synthesis of cyclic AMP (cAMP), activator of PKA. The RAS Associating Domain of Cyr1, containing 2 putative Snf1 phosphorylation sites, was purified in E. coli and its in vitro phosphorylation by Snf1 was demonstrated. Moreover, in a Snf1-G53R strain, in which the kinase is constitutively active, we found a reduction of 50% of intracellular cAMP, together with the deregulation of the expression of PKA-dependent genes. We therefore hypothesized the existence of a crosstalk between the Snf1 and PKA pathways.To investigate the global role of Snf1 in conditions of nutritional sufficiency we performed a transcriptomic analysis (gene chip) of wt and snf1Δ cells grown in 2% and 5% glucose, evidencing that lack of Snf1 causes the deregulation of about 1000 genes in 2%, but not in 5% glucose. Among these there are glycolytic genes and therefore possible metabolic deregulations in the absence of Snf1 were investigated. snf1Δ cells grown in 2% glucose secrete more ethanol and acetate, in proportion to their growth rate, compared to the wt. This enhanced glycolytic activity is abolished, as observed for transcripts, in 5% glucose. We further demonstrated that in our growth condition snf1Δ cells accumulate fatty acids, as previously observed in low glucose, due to the lack of Snf1-dependent phosphorylation of acetyl-CoA carboxylase. An extended metabolic analysis, both through mass spectrometry and NMR, revealed in detail the metabolic rewiring occurring in snf1Δ cells to guarantee the growth in spite of the enhanced anabolic processes. snf1Δ cells in 2% glucose accumulate glutamate, coming from the degradation of supplemented amino acids, in an essential process to maintain the growth rate of the mutant. Moreover, the mutant accumulates TCA cycle intermediates and in 2%, but not 5% glucose, is negatively affected by treatment with antimycin A, inhibitor of the electron transport chain. The treatment impairs growth and ATP content and increases NADH in the mutant, demonstrating the necessity of its mitochondrial reoxidation.

Multiple checkpoint controls ensure that later cellular events are not initiated until previous cellular events have been successfully completed. Our laboratory studies the checkpoint at the G2/M boundary that ensures the integrity of chromosome transmission by blocking mitosis until DNA synthesis and repair is completed. The checkpoint-dependent cell division arrest is one of several prominent responses to DNA damage, which also includes transcriptional induction of damage-inducible genes and DNA repair. I undertook three projects that explore several aspects of the damage response: (1) I further characterized the checkpoint gene RAD24, in that I showed that RAD24 function has G2 phase-specificity after damage and that RAD24 contributes to genomic stability; (2) I evaluated the nature of the damage signal from UV-irradiation that elicits a checkpoint-dependent cell cycle arrest; and (3) I established a transcriptional role for some of the checkpoint genes. In addition, I characterized a gene that encodes a novel elongation factor-type GTPase. Upon examination of the checkpoint-dependent delay following UV-irradiation in a mutant defective for incision of pyrimidine dimers, I found that processing of DNA damage, i.e. dimer-incision, is necessary to generate an appropriate damage signal. Processing of damage may be a general property of the damage response and may involve the checkpoint proteins. I found that some checkpoint genes have an additional role in a complex transcriptional induction response to DNA damage. Primarily, I found: (i) mec1 and mec2 mutants are defective for DNA damage-induction of the RNR3 gene, whereas the other checkpoint mutants appear to play less of a role; (ii) all the checkpoint mutants are proficient for transcriptional induction UBI4; (iii) rad17 mutants, and to a lesser degree mec1 and mec2 mutants as well, are defective for damage-induction of DDR48; (v) transcription of the RAD17, RAD24, MEC1, and MEC2 (but not the RAD9) checkpoint genes is damage-inducible and MEC1 is required for the transcriptional induction of the MEC1 and MEC2 genes, but not the RAD17 or RAD24 genes. I suggest that their transcriptional function ties the checkpoint proteins to DNA repair, as damage-inducible transcriptional induction probably functions to augment repair.

A large fraction of the proteome displays cell cycle-dependent expression, which is important for cells to accurately grow and divide. Cyclical protein expression requires protein degradation via the ubiquitin proteasome system (UPS), and several ubiquitin ligases (E3) have established roles in this regulation. Less is understood about the roles of deubiquitinating enzymes (DUB), which antagonize E3 activity. A few DUBs have been shown to interact with and deubiquitinate cell cycle-regulatory E3s and their protein substrates, suggesting DUBs play key roles in cell cycle control. However, in vitro studies and characterization of individual DUB deletion strains in yeast suggest that these enzymes are highly redundant, making it difficult to identify their in vivo substrates and therefore fully understand their functions in the cell. To determine if DUBs play a role in the cell cycle, I performed a screen to identify specific DUB targets in vivo and then explored how these interactions contribute to cell cycle control. I conducted an in vivo overexpression screen to identify specific substrates of DUBs from a sample of UPS-regulated proteins and I determined that DUBs regulate different subsets of targets, confirming they display specificity in vivo. Five DUBs regulated the largest number of substrates, with Ubp10 stabilizing 40% of the proteins tested. Deletion of Ubp10 delayed the G1-S transition and reduced expression of Dbf4, a regulatory subunit of Cdc7 kinase, demonstrating Ubp10 is important for progression into S-phase. We hypothesized that compound deletion strains of these five DUBs would be deficient in key cellular processes because they regulated the largest number of cell cycle proteins from our screen. I performed genetic analysis to determine if redundancies exist between these DUBs. Our results indicate that most individual and combination deletion strains do not have impaired proliferation, with the exception of cells lacking UBP10. However, I observed negative interactions in some combinations when cells were challenged by different stressors. This implies the DUB network may activate redundant pathways only upon certain environmental conditions. While deletion of UBP10 impaired proliferation under standard growth conditions, I discovered that deletion of the proteasome-regulatory DUBs Ubp6 or Ubp14 rescues the cell cycle defect inubp10∆ cells. This suggests in the absence of Ubp10 substrates such as Dbf4 are rapidly degraded by the proteasome, but deletion of proteasome-associated DUBs restores cell cycle progression. Our work demonstrates that in unperturbed cells DUBs display specificity for their substrates in vivo and that a coordination of DUB activities promotes cell cycle progression.

Le cycle cellulaire eucaryote est caractérisé par des changements abrupts et dynamiques de la polarité cellulaire lorsque les chromosomes sont dupliqués et ségrégés. Ces évènements nécessitent une coordination entre la machinerie du cycle cellulaire et les régulateurs de la polarité. Les mécanismes qui contrôlent cette coordination ne sont pas totalement compris. Dans la levure S. cerevisiae, comme dans d’autres organismes eucaryotes, la GTPase Cdc42 joue un rôle important dans la régulation de la polarité cellulaire. En effet ses régulateurs constituent un module GTPase qui subit une phosphorylation dynamique, au cours du cycle cellulaire, par des kinases évolutivement conservées dont la Cycline-Dependent Kinase 1 (Cdk1) et la p21-Activated Kinase (PAK). Ces kinases et substrats pourraient relier la polarité et la progression dans le cycle cellulaire. En utilisant une approche in vitro, nous avons reconstitué la phospho-régulation du Guanine nucléotide Exchange Factor (GEF) de Cdc42, la protéine Cdc24. Nous avons identifié un possible mécanisme de régulation de la phosphorylation impliquant une protéine d’échafaudage qui augmente la phosphorylation de Cdc24 par la PAK et Cdk1. Cette phosphorylation accroit modérément l’affinité de Cdc24 pour cette même protéine d’échafaudage, Bem1. De plus, en testant les effets d’autres composants du module GTPase sur la phosphorylation de Cdc24, nous avons identifié un effet antagoniste pour une GTPase Activating Protein (GAP), Rga2. Cette protéine est présente dans le même complexe que Cdc24 et Bem1, les membres de ce complexe sont tous phosphorylés par Cdk1. Des mutants rga2 suggèrent que la phosphorylation que subie Rga2 inhibe son activité GAP. Nous proposons un modèle provisoire pour expliquer la présence de Rga2 dans ce complexe et l’inhibition qu’elle oppose à la phosphorylation de Cdc24. La présence de la protéine GAP dans le complexe pourrait être un mécanisme de contrôle de la phosphorylation de Cdc24 dans le but de déstabiliser son intéraction avec la protéine Bem1 en cas de mauvaise localisation du complexe. Par ailleurs, la PAK est activée par l’activité de Cdc42, nos résultats sont consistants avec un modèle dans lequel des signaux du cycle cellulaire engendreraient une auto-amplification de l’activation du module GTPase. Chez S. pombe, la croissance polarisée nécessite un gradient d’activation de Cdc42 dû à une ségrégation de GEF et de GAP. Dans ces travaux nous montrons que toutes les protéines GAPs de Cdc42 localisent aux sites de croissance au cours du cycle cellulaire. Ces localisations sont consistantes avec le besoin de cyclage de Cdc42 pour maintenir sa polarisation. Ces résultats suggèrent que la localisation des protéines GAP régulant Cdc42 chez S. cerevisiae semble différente de ce qui est connu chez S. pombe<br>The eukaryotic cell cycle is characterized by abrupt and dynamic changes in cellular polarity as chromosomes are duplicated and segregated. Those dramatic cellular events require coordination between the cell cycle machinery and polarity regulators. The mechanisms underlying this coordination are not well understood. In the yeast S. cerevisiae, as in other eukaryotes, the GTPase Cdc42 plays an important role in the regulation of cell polarity. Cdc42 regulators constitute a GTPase module that undergoes dynamic phosphorylation during the cell cycle by conserved kinases including Cyclin-Dependent Kinase 1 (Cdk1) and p21-activated kinase (PAK). These kinases and substrates may link cell polarity to the cell cycle progression. Using in vitro approaches, we have reconstituted the phospho-regulation of the Cdc42 Guanine Nucleotide Exchange Factor (GEF), Cdc24. We have identified a possible mechanism of Cdc24 regulation involving a scaffold-dependent increase in Cdc24 phosphorylation by Pak and Cdk1. This phosphorylation moderately increases the affinity of Cdc24 for another GTPase module component, the scaffold Bem1. Moreover, by testing the effect of other GTPase module components on the phosphorylation of Cdc24, and thus on its interaction with the scaffold, we identified an antagonistic function for the GTPase Activating Protein (GAP) Rga2. Our in vivo data of rga2 mutants suggest that Rga2 phosphorylation by Cdk1 inhibits its GAP activity. We propose a tentative model to explain the inhibition of Cla4 by Rga2 and its presence in a complex containing Cdc24 and Bem1. The presence of the GAP protein in the complex may be a mechanism that reduces Cdc24 phosphorylation in case of a mistargetting of the complex in order to downregulate the GEF/Scaffold dimer. Since the PAK component of the GTPase module is itself activated by Cdc42 activity, our results are consistent with a model in which inputs from the cell cycle lead to auto-amplification of the Cdc42 GTPase module. In S. pombe, polarised growth requires a gradient of activation of Cdc42 due to GEF and GAP segregation. Here we show that all Cdc42 GAPs localise to the polarised site during the cell cycle. Those localisations are consistent with a requirement of Cdc42 cycling to maintain a polarity cap. Our results may suggest that Cdc42 GAPs localisations in S. cerevisiae are different from current knowledge in S. pombe

Saccharomyces cerevisiae provides an ideal model to study the regulation of cell cycle commitment due to the high conservation of signalling pathways and regulatory modules through to higher eukaryotes. My work investigates the interplay of cell cycle progression and arrest via the action of transcription factor regulation. Cell cycle commitment is controlled by the cyclin-dependent activation of transcription factor complexes, MBF and SBF. Here I describe the dynamics of SBF and MBF using new polyclonal anti-sera against the three key components Mbp1, Swi4 and Swi6, and their interaction with the inhibitor of SBF, Whi5, and the MBF co-repressor Nrm1. I identify epigenetic modifications that occur on histone proteins at promoters of SBF and MBF genes during the cell cycle. The histone deacetylase Rpd3 has also been investigated as to the role it plays in regulating G1/S transcription. Finally, I have identified a new class of G1/S genes, named switch genes, which are regulated independently by G1/S transcription factors during different phases of the cell cycle. Switch genes are regulated by SBF during G1 and MBF upon entry into S phase, and are enriched for dosage sensitive and replication induced G1/S genes. Switching from SBF-to-MBF allows genes to be activated in response to replication stress, via inactivation of Nrm1. In addition, through switching a potential defect in one of the transcriptional factor complexes will not result in overexpression of these genes. Detailed analysis of the prototypical switch gene TOS4 shows that it is regulated by SBF and MBF, accumulates in response to hydroxyurea, and delays cell cycle progression when over-expressed. The role Tos4 plays in the cell cycle and in response to checkpoint activation remains unclear, however, data suggests a role in modulating HDAC activity. The roles other switch genes play in response to checkpoint activation are yet to be investigated.

Rho GTPases are highly conserved regulators of cell polarity and the actin cytoskeleton. The role of the Rho GTPase Cdc42 and its regulation during cell division is not well understood. Using biochemical and imaging approaches in budding yeast, I demonstrate that Cdc42 activation peaks during the G1/S transition and during anaphase, but drops during mitotic exit and cytokinesis. Cdc5/Polo kinase is an important upstream cell cycle regulator that suppresses Cdc42 activity. Failure to downregulate Cdc42 during mitotic exit prevents the normal localization of key cytokinesis regulators - Iqg1 and Inn1- at the division site, and results in an abnormal septum. The effects of Cdc42 hyperactivation are largely mediated by the Cdc42 effector p21-activated kinase (PAK) kinase, Ste20. Inhibition of Cdc42 and related Rho GTPases may be a general feature of cytokinesis in eukaryotes.

The budding yeast cell cycle has attracted attention from many experimentalists over the years for its simplicity and amenability to genetic manipulation. Moreover, the regulatory components described in budding yeast, Saccharomyces cerevisiae, are conserved in higher eukaryotes. The budding yeast cell cycle is governed by a complex network of chemical reactions controlling the activity of the cyclin-dependent kinases (CDKs), proteins that drive the major events of the cell cycle. The presence of these proteins is required for the transition from G1 to S phase (Start) whereas their absence permits the transition from S/M to G1 phase (Finish). The cell cycle of budding yeast is based on alternation between these two states. To test the accuracy of this theory against experiments, we built a hypothetical molecular mechanism of the budding yeast cell cycle and transcribed it into differential equations. With a proper choice of kinetic parameters, the differential equations reproduce the main events of the cell cycle such as: the synthesis of cyclins (Cln1,2; Cln3; Clb1,2; Clb5,6) by their transcription factors (SBF, Mcm1, MBF); their association with stoichiometric inhibitors (Sic1, Cdc6); their degradation by SCF and adaptors of the APC (Cdc20, Cdh1). The emphasis was put on mechanisms responsible for the release of Cdc14 from the RENT complex, Cdc14 being a major player in exit from mitosis. Simulations of the wild type strain and more than 100 mutants showed phenotypes in accordance with experimental observations. Some mutants defective in the Start and Finish transitions and the different ways to rescue them will be presented.<br>Ph. D.

Control of cell cycle by Stress Activated Protein Kinases (SAPKs) is an essential aspect for adaptation to extracellular stimuli. In Saccharomyces cerevisiae, the activation of the Hog1 SAPK, results in a delayed transcription of the G1 cyclins CLN1,2 and the stabilization of the B-type cyclin inhibitor SIC1, therefore postponing entry into S phase. The results displayed here, show, by a combination of mathematical modelling and quantitative in vivo experiments, that, before Start, the control of G1-S transition is mainly exerted by inhibiting expression of cyclins, both G1 (CLN1,2) and S phase (CLB5,6) cyclins. On the other hand, after Start, it is the phosphorylation and stabilization of Sic1 by Hog1 that becomes imperative to prevent inadequate firing of replication before adaptation. Therefore, we found that there is a distinct temporal role for Sic1 and cyclins on the G1 regulation by a SAPK in response to stress. We have also found that Hog1 induces a G2 delay, by down-regulating CLB2 transcription and phosphorylating Hsl1 to promote Hsl7 delocalization and subsequent accumulation of Swe1, an inhibitor of Clb1,2-Cdc28, and thus postponing anaphase. Altogether, we demonstrate novel Systems Biology approaches are useful to better understand how an intracellular signalling pathway incises on cell cycle control, beyond a mechanistic description, as well as showing how a single MAPK modulates different cell cycle checkpoints to improve cell survival upon stress.<br>El control del cicle cel·lular per Proteïna Cinases Activades per Estrès (SAPKs) es un aspecte essencial per a l'adaptació als estímuls extracel·lulars. A Saccharomyces cerevisiae, l'activació de la SAPK Hog1, resulta en un retardament de la transcripció de les ciclines de G1 (CLN1,2) i l'estabilització del inhibidor de les ciclines del tipus B, SIC1, i per tant posposa l'entrada en fase S. Els resultats que aquí s'exposen, mostren, mitjançant la combinació de modelatge matemàtic i experiments quantitatius in vivo, que, abans d'Start, el control de la transició es duu a terme principalment inhibint l'expressió de les ciclines, tant les de G1 (CLN1,2) com les de la fase S (CLB5,6). Per altra banda, després d'Start, la fosforilació i estabilització de Sic1 per part de Hog1 esdevé un fet necessari per a prevenir la iniciació de la replicació abans d'adaptar-se. Per tant, hem descobert aquí que la regulació de Sic1 i les ciclines juguen un paper diferent segons el moment en que apareix l'estrès. Hem descrit també, que Hog1 produeix una parada a G2 a través de la inhibició de la transcripció de CLB2 i la fosforilació d'Hsl1, la qual promou la deslocalització d'Hsl7 i la subsegüent estabilització de Swe1, un inhibidor específic de Clb1,2-Cdc28, i d'aquesta manera es posposa l'entrada en Anafase. Tot plegat, demostra que l'ús d'aproximacions pròpies de la Biologia de Sistemes és útil per a entendre de quina forma una via de senyalització intracel·lular incideix sobre el control del cicle cel·lular, mes enllà de la pura descripció de la mecànica del sistema. D'aquesta forma, proposem que una sola MAPK modula distints punts de control del cicle cel·lular per millorar la probabilitat de supervivència en front de l'estrès osmòtic.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2003.<br>Includes bibliographical references.<br>Advances in genome sequencing and comparative genomics have uncovered ancient duplications in the genomes of many extant organisms. Evidence for large regional duplications is observed in eukaryotic organisms that include yeast, plants, fish, and humans. Furthermore, phylogenetic analysis of paralogous duplications within these organisms provides support for a single duplication event of the entire genome. The prevalence of genomic duplications lends credence to proposals that suggest that evolution is driven by polyploidization. This evidence must be balanced by recent experiments that demonstrate that newly formed polyploid genomes manifest problems in genomic stability, gene regulation, and development. In order to determine the consequences of nascent duplications of the entire genome, I created isogenic polyploid strains in Saccharomyces cerevisiae. These newly formed polyploids do not grow abnormally during exponential growth. Furthermore, they are not increased or decreased in their sensitivity to a variety of stresses including oxidative stress, high osmolarity, salt stress, toxic ions, and growth at high temperatures. However, polyploid strains of S. cerevisiae rapidly lose viability under conditions of nutrient deprivation. In contrast to isogenic haploids that remain viable for weeks and even months, tetraploid yeast are completely inviable after approximately 10-15 days in synthetic media. Analysis of the growth patterns of haploid and tetraploid cells during stationary phase reveals that tetraploids are defective for growth arrest during nutrient deprivation.<br>(cont.) Furthermore, alterations that impede their inappropriate mitotic growth, such as deletion of the G1 cyclin, CLN3, can restore viability in tetraploids during stationary phase. The stationary phase defects found in tetraploid cells are notably similar to those observed in haploid cells that constitutively activate the glucose sensing Ras/cAMP pathway. In addition, all of these defects are suppressed by overexpression of the Ras/cAMP pathway inhibitor, RPII. Although these data suggest a role for RPIH in the restoration of tetraploid viability, the precise function remains elusive. Nevertheless, RPI1 may define a compensatory change that permits the survival of nascent polyploid organisms.<br>by Alexis Albert Andalis.<br>Ph.D.

In this study, we used budding yeast as a model organism to examine the effects of overexpression of Bmh1, a yeast homolog of 14-3-3γ. We found that in the presence of modest DNA damage, Bmh1 overexpression had its most prominent effect during G2/M-phase of the cell cycle. We also observed that overexpression of Bmh1 concurrent with the induction of DNA damage partially rescued the G2/M arrest defect caused by the absence of Rad9, a key component of the G2/M DNA damage checkpoint pathway. When RAD53, a gene in the "Rad53 pathway" of the G2/M checkpoint, was deleted, overexpression of Bmh1 had no effect. However, overexpression of Bmh1 in a strain bearing the rad53-11 mutation partially rescued the arrest. Additionally, Bmh1 overexpression had a minimal effect on the G2/M arrest response with deletion of Chk1, a key component of the parallel G2/M checkpoint pathway. This led us to hypothesize that overexpression of Bmh1in the absence of Rad9 modulates the Rad53 pathway. We propose a model in which the rescue of Rad9’s otherwise obligatory role in the DNA damage checkpoint is the consequence of Bmh1 subserving the adaptor function of Rad9 by bringing Mec1 and Rad53 together.

Glucose and regulation of cell cycle in S. cerevisiae: analysis of mutants impaired in sugar uptake mechanisms Requisito fondamentale per la sopravvivenza di microrganismi a vita libera come il lievito S. cerevisiae è la capacità di regolare il proprio metabolismo e la progressione del ciclo cellulare in modo tale che la crescita sia rapida in presenza di abbondanti nutrienti e si arresti all’esaurirsi degli stessi. Perché questo sia possibile, nutrienti come il glucosio devono generare segnali che vengano recepiti ed elaborati dal complesso macchinario che governa il ciclo cellulare. S. cerevisiae possiede almeno tre meccanismi per rilevare variazioni dei livelli di glucosio nel mezzo di coltura: il pathway di Rgt2/Snf3, che controlla l’espressione dei trasportatori degli zuccheri esosi; il pathway cAMP/PKA, che regolando l’attività della protein-kinasi A promuove l’espressione di geni coinvolti nel metabolismo fermentativo e nella crescita cellulare e inibisce la trascrizione di geni coinvolti nella risposta agli stress; il glucose main repression pathway, che reprime l’espressione di geni coinvolti nella respirazione cellulare, nella gluconeogenesi e nell’utilizzo di fonti di carbonio alternative al glucosio. L’assunzione di glucosio nel citoplasma dall’ambiente esterno avviene attraverso i trasportatori codificati dalla famiglia di geni HXT (HeXose Transporter), che comprende almeno 20 membri: HXT1-17, RGT2, SNF3 e GAL2. Snf3 e Rgt2 sono incapaci di trasportare lo zucchero, ma agiscono piuttosto da sensori del livello di glucosio extracellulare: in particolare, Snf3 rileva basse concentrazioni dello zucchero inducendo l’espressione dei trasportatori ad alta affinità (codificati dai geni HXT2-HXT4), mentre Rgt2 rivela alte concentrazioni di glucosio promuovendo l’espressione dei trasportatori a bassa affinità (HXT1). Nessuno dei trasportatori è essenziale e solo la delezione di tutti i geni HXT (o almeno di quelli compresi tra 1-7 in alcuni background) rende la cellula di lievito incapace di crescere in presenza di glucosio come unica fonte di carbonio. L’espressione dei vari trasportatori è regolata a livello trascrizionale attraverso un complesso network che coinvolge tutti e tre pathway deputati al sensing del glucosio: come risultato, S. Cerevisiae è in grado di mantenere sempre un alto flusso glicolitico esprimendo il set di trasportatori più adatto alla quantità di glucosio disponibile. Le connessioni tra i pathway deputati al sensing del glucosio e gli elementi di regolazione del ciclo cellulare non sono completamente definite, anche perché risulta spesso difficile scindere il duplice ruolo dello zucchero come nutriente e come molecola segnale. Obbiettivo del presente progetto di ricerca è chiarire gli effetti di alterazioni nei meccanismi di sensing e (in modo particolare) di trasporto del glucosio sulla coordinazione tra crescita e divisione cellulare. In una prima fase dello studio sono stati presi in esame alcuni mutanti con delezioni nei geni HXT1-7, codificanti per i principali trasportatori degli zuccheri esosi: i dati presenti in letteratura certificano infatti come queste mutazioni siano sufficienti ad abolire sostanzialmente l’assunzione (uptake) cellulare del glucosio impedendo la crescita dei mutanti su tale fonte di carbonio. I parametri di crescita (tempo di duplicazione, indice di gemmazione, contenuto proteico e di DNA) di ciascuno dei ceppi sono stati determinati in due condizioni sperimentali: i) crescita esponenziale bilanciata in terreno csm/YNB addizionato di 2% etanolo o di una miscela 2% Etanolo + 2% glucosio, da cui è emerso come il glucosio possa esercitare un effetto sulle dimensioni celulari anche nei mutanti hxt (indipendente quindi dal suo ruolo come nutriente). Infatti, analogamente alle cellule wild-type, anche i ceppi con i trasportatori deleti mostrano dimensioni cellulari e contenuto proteico maggiore quando fatti crescere in glucosio+Etanolo, sebbene (diversamente dal ceppo wild type) la loro velocità di crescita sia simile a quella registrata in terreno con solo etanolo; ii) crescita durante shift-up nutrizionale etanolo => glucosio. All’aggiunta dello zucchero le cellule wild-type vanno incontro ad fase iniziale di adattamento (evidenziato dalla diminuzione transiente (10-15%) dell’indice di gemmazione), necessaria per reimpostare profilo trascrizionale, velocità di crescita e dimensioni cellulari e per il successivo passaggio ad un metabolismo energetico di tipo fermentativo. A differenza del ceppo wild type, dopo l’aggiunta di glucosio le cellule hxt(1-7) manifestano una drammatica e prolungata riduzione nell’indice di gemmazione e un forte rallentamento (arresto) nella progressione del ciclo cellulare. In seguito le cellule riprendono a dividersi con una velocità sostanzialmente identica a quella precedente lo shift, mentre volume cellulare e contenuto proteico medio aumentano sensibilmente: l’effetto del glucosio sulle dimensioni cellulari dei mutanti hxt(1-7) è tuttavia transiente e si esaurisce nell’arco di due/tre round di divisioni, quando le cellule tornano ad assumere le dimensioni tipiche della crescita su etanolo. I dati finora riportati sembrano quindi suggerire che, almeno inizialmente, gli effetti del glucosio sulle dimensioni cellulari dipendano dal sensing dello zucchero e non dal suo metabolismo. Tuttavia, sebbene il ceppo hxt(1-7) non sia in grado di crescere su glucosio come unica fonte, rimane comunque dotato di una capacità residua di trasporto dello zucchero, che sebbene insufficiente a sostenere il metabolismo glicolitico, potrebbe comunque assumere un’importanza decisiva per la regolazione delle dimensioni cellulari. Nel tentativo di scindere ancora più nettamente il duplice ruolo del glucosio come nutriente e come molecola segnale, in una successiva fase di studi sono stati utilizzati mutanti con delezioni in tutti i geni per i trasportatori del glucosio (hxt(1-17)), in cui ogni residuo trasporto dello zucchero risulta abolito. In aggiunta, si sono presi in esame una serie di mutanti con una capacità di uptake del glucosio progressivamente ridotta: nel dettaglio, la lista comprende (oltre ovviamente al ceppo wild type di riferimento): i) hxt(1-17)gal2, in cui il trasporto del glucosio è completamente abolito; ii) il ceppo hxt(1-17)) snf3, in cui l’inattivazione del sensore SNF3 rispristina una trascurabile capacità di trasporto del glucosio, insufficiente comunque a garantire la crescita in terreno liquido contenente glucosio come unica fonte: l’assunzione dello zucchero in questo caso sembrerebbe avvenire attraverso un trasportatore non ancora caratterizzato, la cui trascrizione risulta derepressa in assenza di SNF3; iii) il ceppo (hxt(1-17) gal2 HXT1, che esprime in modo costitutivo come unico trasportatore HXT1, un carrier a bassa affinità; iv) ) il ceppo (hxt(1-17) gal2 HXT7, che esprime in modo costitutivo come unico trasportatore HXT7, un carrier ad alta affinità; v) il ceppo snf3 rgt2, in cui l’uptake del glucosio è ridotto a causa dell’inattivazione del principale pathway che regola l’espressione dei maggiori trasportatori; vi) il triplo deleto hxk2 hxk1 glk1, che è in grado di trasportare glucosio nel citoplasma ma non è in grado di metabolizzarlo a causa dell’assenza di tutte e tre le chinasi che catalizzano il primo passaggio della glicolisi. I ceppi sopra elencati sono stati sottoposti alle analisi descritte in precedenza. Nel caso dei ceppi capaci di metabolizzare il glucosio, il tasso di crescita e le dimensioni cellulari su tale fonte sono generalmente correlate all’efficienza del sistema di trasporto dello zucchero nei vari mutanti: sembra esistere una relazione sostanzialmente lineare tra velocià di consumo del glucosio/velocità di crescita/dimensioni cellulari. Unica eccezione pare essere il ceppo snf3 rgt2, che manifesta dimensioni cellulari notevolmente più ridotte rispetto a quanto atteso sulla base del suo tasso di crescita: un risultato che sembrerebbe suggerire un ruolo diretto del pathway Snf3/Rgt3 nei meccanismi che regolano le dimensioni cellulari in risposta ai nutrienti. Diversamente da quanto emerso in precedenza, la crescita dei mutanti hxt(1-17) risulta fortemente inibita in terreni contenenti miscele di etanolo (o altra fonte non fermentabile) e glucosio, anche quando la concentrazione dello zucchero è a livelli sub-ottimali (0.05% anziché 2%). L’aggiunta di glucosio a cellule hxt(1-17) in crescita su etanolo (shift-up nutrizionale) determina l’arresto permanente del ciclo cellulare in G1 (cellule vitali, non gemmate con contenuto di DNA presintetico). Sembra quindi che la semplice presenza di glucosio nell’ambiente extracellulare - ma non il trasporto dello zucchero nel citoplasma - sia sufficiente ad impedire l’utilizzo di fonti di carbonio alternative presenti nel medium: ciò spiegherebbe la mancata crescita in terreni misti glucosio+etanolo da parte di cellule hxt(1-17), incapaci di effettuare l’uptake dello zucchero. Se tale ipotesi fosse corretta, inattivando contemporaneamente tutti i pathway deputati al sensing del glucosio dovrebbe essere possibile ripristinare la crescita di cellule hxt(1-17) in terreni contenenti miscele di glucosio ed etanolo. Al momento, si è appurato che l’inattivazione del ramo del cAMP/PKA pathway passante attraverso Gpr1/Gpa2 non è sufficiente a correggere il difetto di crescita del ceppo hxt(1-17)gal2 in fonte mista glucosio/etanolo. Al contrario, la semplice inattivazione di SNF3 (ma non di RGT2)sembra sostanzialmente azzerare l’effetto citostatico del glucosio sulla crescita del ceppo hxt(1-17) gal2 snf3. L’interpretazione di tale risultato è ovviamente complicata dal fatto che la delezione di SNF3 ripristina parzialmente il trasporto del gluccosio in un ceppo privo di tutti i trasportatori, sebbene, vale la pena ricordare, su scala estremamente ridotta e comunque insufficiente a sostenere la crescita, come confermato attraverso misurazioni dirette della velocità di consumo delo zucchero nel ceppo hxt(1-17)) snf3. Tuttavia, diversi dati in letteratura suggeriscono come il pathway Snf3/Rgt2 partecipi in qualche misura ai meccanismi della glucose repression, in particolare attrverso Mig2, un repressore trascrizionale che in presenza di glucosio collabora con Mig1 nel reprimere la trascrizione di geni richiesti per l’utilizo di fonti di carbonio alternative. La delezione di MIG2 non è tuttavia sufficiente a ripristinare la crescita su etanolo/glucosio del ceppo hxt(1-17)gal2: ulteriori indagini sono dunque necessarie per chiarire quale sia il ruolo giocato da SNF3 nell’intero processo. In aggiunta, il comportamento manifestato dal ceppo hxk2 hxk1 glk1 durante shift-up nutrizionale da etanolo a glucosio sembra ulteriormente confermare come in lievito lo zucchero sia in grado di regolare le dimensioni cellulari indipendentemente dal proprio metabolismo, almeno in una fase iniziale: le cellule hxk2 hxk1 glk1 in crescita su etanolo rispondono all’aggiunta di glucosio aumentando considerevolmente il proprio volume, in misura paragonabile a quanto si registra nel ceppo wild type; tuttavia, contrariamente al ceppo wild type, nel mutante hxk2 hxk1 glk1 l’aumento delle dimensioni celluari si accompagna ad un progressivo rallentamento della velocità di crescita, fino ad un totale arresto del ciclo di divisione cellulare che sopraggiunge a circa 12 ore dallo shift. Dopo una fase di lag piuttosto prolungata ed estremamente variabile, in cui le cellule, pur non dividendosi, si mantengono gemmate, si assiste alla rispresa del ciclo di divisione cellulare: le cellule tornano a dividersi lentamente utilizzando l’etanolo residuo nel terreno e nell’arco di due/tre generazioni assumono nuovamente le tipiche dimensioni ridotte associate alla crescita su fonte di carbonio non fermentabile. Ad ulteriore conferma di come l’effetto del glucosio sia solo temporaneo, dimensioni e contenuto proteico di cellule hxk2 hxk1 glk1 in crescita bilanciata su etanolo o su fonte mista etanolo/glucosio sono sostanzialmente identiche. Nonostante il sorprendente effetto citostativo dello zucchero, l’aumento delle dimensioni cellulari in risposta all’aggiunta di glucosio si manifesta anche nel ceppo privo di tutti i trasportatori (hxt(1-17) gal2)., sebbene in misura meno eclatante rispetto al triplo mutante hxk2 hxk1 glk1. Nell'insieme, tali risultati sembrano confermare come il glucosio sia in grado di modulare le dimensioni della cellula di lievito in maniera (almeno in parte) indipendente dal proprio ruolo come nutriente, funzionando in buona sostanza come un "ormone". Per chiarire le basi molecolari di tale fenomeno è necessario chiarire le connessioni tra i pathway deputati al sensing del glucosio e gli elementi di regolazione del ciclo di divisione cellulare in S. cerevisiae. A tal fine, si è ultimata la costruzione di una serie di mutanti esprimenti versioni “taggate” (HA-tag) di alcuni dei principali regolatori coinvolti nella transizione G1/S (nello specifico Cln3, Cln2, Far1, Sic1 e Clb5), così da facilitare l’analisi dei loro livelli di espressione e di localizzazione subcellulare. Gli studi in questo senso sono tuttavia in una fase ancora troppo preliminare per poter trarre conclusioni definitive. Da utlimo, si è cercato di valutare il contributo relativo di sensing, trasporto e metabolismo del glucosio alla regolazione trascrizionale del gene SUC2, uno dei marcatori più comunemente utilizzati per valutare il fenomeno della glucose repression in S. cerevisiae. SUC2 codifica per l’invertasi, un enzima chiave per l’utilizzo del disaccaride saccarosio e la sua espressione risulta completamente bloccata in presenza di alti livelli di glucosio mentre viene indotta da raffinosio o da bassi livelli di glucosio. I risultati ottenuti con i vari mutanti hanno evidenziato come in presenza di abbondante glucosio il livello basale di attività invertasica sia generalmente proporzionale alla velocità del flusso glicolitico, che dipende in larga misura dalla capacità di trasporto dello zucchero: nei ceppi aventi un sistema di uptake per il glucosio ad efficienza ridotta l’invertasi risulta parzialmente o addirittura competamente derepressa, come nel caso del mutante privo di tutti i trasportatori. Il ceppo snf3 rgt2 sfugge invece a questa regola, in quanto l’attività invertasica risulta sì parzialmente derepressa in presenza di alte concentrazioni di glucosio, ma non più inducibile da bassi livelli di glucosio o raffinosio. In aggiunta, l’inattivazione di SNF3 e di RGT2 abolisce completamente l’induzione dell’attività invertasica nel ceppo hxt(1-17)gal2 in presenza di glucosio. Nell’insieme, i dati appena descritti sembrano suggerire per il pathway Snf3/Rgt2 un ruolo decisamente più rilevante nella regolazione di SUC2 rispetto a quanto gli viene comunemente attribuito. Esperimenti futuri permetteranno di chiarire meglio la questione.

Der Zellzyklus organisiert die Zellteilung, und kontrolliert die Replikation der DNA sowie die Weitergabe des Genoms an die nächste Zellgeneration. Er unterliegt einer strengen Kontrolle auf molekularer Ebene. Diese molekularen Kontrollmechanismen sind für das Überleben eines Organismus essentiell, da Fehler Krankheiten begüngstigen können. Vor allem Krebs ist assoziiert mit Abweichungen im Ablauf des Zellzyklus. Die Aufklärung solcher Kontrollmechanismen auf molekularer Ebene ermöglicht einerseits das Verständnis deren grundlegender Funktionsweise, andererseits können solche Erkenntnisse dazu beitragen, Methoden zu entwickeln um den Zellzyklus steuern zu können. Um die molekularen Abläufe des Zellzyklus in ihrer Gesamtheit besser zu verstehen, eignen sich computergestützte Analysen. Beim Zellzyklus handelt es sich um einen Signaltransduktionsweg. Die Eigenschaften dieser Prozesse stellen Rekonstruktion und Übersetzung in digital lesbare Formate vor besondere Herausforderungen in Bezug auf Skalierbarkeit, Simulierbarkeit und Parameterschätzung. Diese Studie präsentiert eine großskalige Netzwerkrekonstruktion des Zellzyklus des Modellorganismus Saccharomyces cerevisiae. Hierfür wurde die reaction-contingency Sprache benutzt, die sowohl eine mechanistisch detaillierte Rekonstruktion auf molekularer Ebene zulässt, als auch deren Übersetzung in ein bipartites Boolesches Modell. Für das Boolesche Modell mit 2506 Knoten konnte ein zyklischer Attraktor bestimmt werden, der das Verhalten einer sich teilenden Hefezelle darstellt. Das Boolesche Modell reproduziert zudem das erwartete phänotypische Verhalten bei Aktivierung von vier Zellzyklusinhibitoren, und in 32 von 37 getesteten Mutanten. Die Rekonstruktion des Zellzyklus der Hefe kann in Folgestudien genutzt werden, um Signaltransduktionswege zu integrieren, die mit dem Zellzyklus interferieren, deren Schnittstellen aufzuzeigen, und dem Ziel, die molekularen Mechanismen einer ganzen Zelle abzubilden, näher zu kommen. Diese Studie zeigt zudem, dass eine auf reaction- contingency Sprache basierte Rekonstruktion geeignet ist, um ein biologisches Netzwerk konsistent mit empirischer Daten darzustellen, und gleichzeitig durch Simulation die Funktionalität des Netzwerkes zu überprüfen.<br>The survival of a species depends on the correct transmission of an intact genome from one generation to the next. The cell cycle regulates this process and its correct execution is vital for survival of a species. The cell cycle underlies a strict control mechanism ensuring accurate cell cycle progression, as aberrations in cell cycle progression are often linked to serious defects and diseases such as cancer. Understanding this regulatory machinery of the cell cycle offers insights into how life functions on a molecular level and also provides for a better understanding of diseases and possible approaches to control them. Cell cycle control is furthermore a complex mechanism and studying it holistically provides for understanding its collective properties. Computational approaches facilitate holistic cell cycle control studies. However, the properties of the cell cycle control network challenge large-scale in silico studies with respect to scalability, model execution and parameter estimation. This thesis presents a mechanistically detailed and executable large-scale reconstruction of the Saccharomyces cerevisiae cell cycle control network based on reaction- contingency language. The reconstruction accounts for 229 proteins and consists of three individual cycles corresponding to the macroscopic events of DNA replication, spindle pole body duplication, and bud emergence and growth. The reconstruction translated into a bipartite Boolean model has, using an initial state determined with a priori knowledge, a cyclic attractor which reproduces the cyclic behavior of a wildtype yeast cell. The bipartite Boolean model has 2506 nodes and correctly responds to four cell cycle arrest chemicals. Furthermore, the bipartite Boolean model was used in a mutational study where 37 mutants were tested and 32 mutants found to reproduce known phenotypes. The reconstruction of the cell cycle control network of S. cerevisiae demonstrates the power of the reaction-contingency based approach, and paves the way for network extension with regard to the cell cycle machinery itself, and several signal transduction pathways interfering with the cell cycle.

High fidelity chromosome segregation in all cells requires the formation of bi-oriented attachments between spindle microtubules (MT) and chromosomes. The kinetochore provides a bridge between the MTs and chromosomes. Ame1 is an essential but undercharacterized component of the central kinetochore COMA sub-complex (Ctf19, Okp1, Mcm21, Ame1). In order to characterize Ame1, I used two conditional alleles of the COMA, ame1-4 and okp1-5. I examined the role of Ame1 in the context of the kinetochore and in the maintenance of the spindle assembly checkpoint (SAC) and the formation and repair of kinetochore-MT attachments. I found that ame1-4 cells have a compromised COMA. In contrast, the COMA is disrupted in okp1-5 cells, but Ame1 remains localized to the kinetochore. Nonetheless, the stability of DNA binding and MT binding kinetochore complexes remains intact in ame1-4 and okp1-5 cells. I used the difference between okp1-5 and ame1-4 to further delineate the relationship between Ame1 and the COMA. ame1-4 cells exhibit defective sister chromatid attachments that are not repaired, and are unable to maintain a checkpoint arrest. We find that disruption of the COMA results in a failure to maintain the localization of Sli15 to the kinetochore. In turn, Sli15 functions in checkpoint maintenance and spindle passenger protein migration. Indicative of the loss of passenger protein migration, ame1-4 cells exhibit a cytokinesis defect. Finally, over-expression of OKP1 in ame1-4 cells restores localization of ame1-4p and re-establishes checkpoint maintenance but does not restore Sli15 kinetochore localization. A synthetic genetic screen (SGA) was carried out to identify genetic interactors of the ame1-4 allele. Thirty-three (33) genes were found to interact with ame1-4 to produce a synthetic sick/lethal phenotype. Many genes identified were common interactors with other kinetochore subunits. Comparing the genetic interaction network of the COMA genes, ame1-4 shared many interac<br>Dans toute cellule en division, la haute fidélité de la ségrégation des chromosomes passe par la formation d'attachements bi orientés entre les microtubules du fuseau (MT) et les chromosomes. Le kinétochore est la structure faisant le lien entre les MT et les chromosomes. Le complexe COMA (Ctf19, Okp1, Mcm21, Ame1) est un sous-complexe central du kinétochore, et Ame1 en est un composant essentiel quoique peu caractérisé. Dans le but de caractériser Ame1, j'ai utilisé deux allèles du complexe COMA: ame1-4 et okp1-5. J'ai examiné le rôle d'Ame1 dans le cadre du kinétochore et de la maintenance du point de contrôle de l'assemblage du fuseau ainsi que dans la formation et la réparation des attachements entre kinetochore et MT. J'ai constaté que, dans les cellules ame1-4, le complexe COMA est compromis. Dans les cellules okp1-5, le complexe COMA est également perturbé, alors qu'Ame1 est localisé au kinétochore. Toutefois, dans les cellules ame1-4 comme dans les cellules okp1-5, la stabilité de la liaison à l'ADN et aux MT des complexes du kinétochore demeure intacte. J'ai utilisé la différence entre les mutants okp1-5 et ame1-4 pour mieux comprendre la relation entre Ame1 et le complexe COMA. Dans les cellules ame1-4, les attachements des chromatides soeurs sont déficients. Ceux-ci ne sont pas réparés et sont incapables de maintenir l'arrêt de croissance suite à l'activation du point de contrôle. Nous avons constaté que l'inactivation du complexe COMA provient de l'incapacité à localiser Sli15 au kinétochore. Par ailleurs, Sli15 joue un rôle dans la maintenance du point de contrôle et la migration des protéines passagères du fuseau. Le fait que les cellules ame1-4 présentent une cytokinèse déficiente indique une perte de migration des protéines passagères. Enfin, la surexpression d'OKP1 dans les cellules ame1-4 rétablit la localisation de ame1-4 et la maintenance du point de contrôle mais pas la localisation de Sli15 au$

Exposure of yeast to increases in extracellular osmolarity activates the stress-activated Hog1 MAP kinase, which is essential for cell survival upon osmotic stress. Activation of the Hog1 MAPK results in cell growth arrest, suggesting a possible role of the MAP kinase in the control of the cell cycle. Our results have shown that Hog1 activation resulted in accumulation of cells in the G1/S and G2/M transitions. At G1, Hog1 regulates the cell cycle progression by a dual mechanism that involves downregulation of G1 cyclin expression and direct targeting of the CDK-inhibitor protein Sic1. The MAPK interacts with Sic1, and phosphorylates a single residue of Sic1, which, in combination with the downregulation of cyclin expression, results in Sic1 stabilization and inhibition of cell cycle progression. Consistently, sic1_ cells, or cells containing a SIC1 allele mutated in the Hog1 phosphorylation site, are unable to arrest at G1 phase after Hog1 activation, and become sensitive to osmostress. Together, our data indicate that Sic1 is the molecular target for Hog1 that is required to modulate cell cycle progression in response to stress at G1. On the other hand, activation of the Hog1 MAPK also results in an increase of cells in the G2 phase. Arrested cells displayed down regulation of the Clb2-Cdc28 kinase activity and consequently enlarged buds, defects in spindle formation and orientation. These effects were prevented by deletion of the SWE1 gene. Thus, swe1Ä cells failed to arrest at G2, which resulted in a premature entry into mitosis and mislocalization of nuclei. Consistently, swe1Ä cells were osmosensitive. Swe1 degradation was reduced in response to activation of Hog1. Swe1 accumulation is mediated by the activity of the complex Hsl1-Hsl7. Hog1 phosphorylates a single residue at the regulatory domain of Hsl1, which leads to the mislocalization of Hsl7 from the bud neck, and consequent Swe1 accumulation. In addition, Hog1 downregulates G2 cyclin expression, reinforcing the inhibition of cell cycle progression at G2/M. These results indicate that Hog1 imposes a delay in critical phases of cell cycle progression necessary for proper cellular adaptation to new extracellular conditions.

The decision to initiate division is very important, as once cells have initiated division they are committed to complete it. In Saccharomyces cerevisiae, commitment to a new round of cell division occurs at a regulatory point in late G1 called START. Progression through START requires the activation of the cyclin dependent kinase Cdc28p by the G1 cyclins. G1 cyclins in complex with Cdc28p activate the transcription of approximately 100 genes involved in the G1 to S transition and degradation of Sic1p, an inhibitor of B type cyclins, and thus are important for initiation of DNA replication. Despite the widely studied role of regulatory cyclins and cyclin dependent kinase in the G1 to S transition, how cells determine when to initiate DNA replication is poorly understood. We have identified several gene products, which when overexpressed, cause cells to initiate DNA replication faster than wild type. Here we discuss the role of DCR2 (Dosage dependent Cell cycle Regulator), GID8 (Glucose Induced Degradation) and KEM1 (Kar-Enhancing Mutation) in the regulation of START. Over expression of DCR2 and GID8 accelerates initiation of DNA replication. Cells lacking both these genes delay initiation of DNA replication. Genetic analysis suggests that Gid8p functions upstream of Dcr2p to promote START. Further, we show that DCR2, which codes for a metallo-phosphoesterase, might regulate completion of START by affecting degradation of Sic1p. Over expression of DCR2 lowers the half-life of Sic1p without altering the expression of Cln2p. The evidence suggests that Dcr2p affects START completion through dephosphorylation of Sic1p. KEM1 is a Saccharomyces cerevisiae gene, conserved in all eukaryotes, which codes for a 5’-3’ cytoplasmic exonuclease. This exonuclease is involved in exiting mitosis, by degrading the mRNA of the mitotic cyclin CLB2. Besides its role in mitotic exit, an enzymatically inactive version of Kem1p can accelerate the G1 to S transition and initiation of DNA replication when over expressed. This result suggests that Kem1p might have a previously unrecognized role in the G1 to S transition independent of its exonuclease activity, and supports the notion that Kem1p is a multifunctional protein with distinct and separable roles.

La cohésine est un complexe protéique conservé dans l'évolution composé d'un anneau capable d'embrasser l'ADN et de protéines auxiliaires régulant son association avec l'ADN. D'une part, la cohésine confère la cohésion des chromatides sœurs nécessaire à leur ségrégation, d'autre part elle établit et maintient des boucles de chromatine. Ces boucles sont requises pour la formation de domaines topologiques, l'expression génique et la stabilité du génome. Cependant les mécanismes régissant leur formation ne sont pas entièrement élucidés. Selon le modèle d'extrusion de boucles, la cohésine capturerait des boucles de petites tailles et les élargirait en extrudant l'ADN à travers son anneau. Dans ce modèle, la taille des boucles dépendrait à la fois du temps de résidence des cohésines sur l'ADN et de leur processivité. Étudier la régulation des cohésines est donc fondamental pour comprendre la biologie des chromosomes. Dans cette étude nous avons montré que les bras des chromosomes mitotiques de la levure Saccharomyces cerevisiae étaient organisés sous forme de boucles de chromatine dépendantes des cohésines. Nous avons étudié le rôle des sous-unités régulatrices des cohésines, Pds5, Wpl1 et Eco1 dans la formation de ces boucles. Nos données montrent que Pds5 inhibe leur expansion, via Wpl1 et Eco1. Comme décrit chez les mammifères, Wpl1 les abolit en dissociant les cohésines des chromosomes. En revanche, nos résultats suggèrent qu'Eco1 entraverait la translocation des cohésines sur l'ADN, nécessaire pour l'agrandissement des boucles. Nous avons ensuite analysé le rôle de ces protéines dans l'organisation de l'ADN ribosomique (ADNr), séquence enrichie en cohésines, hautement transcrite et isolée du reste du génome. Pds5 semble avoir un rôle central dans l'organisation de cette séquence, qui ne dépendrait pas de Wpl1 ou d'Eco1. Afin d'analyser de manière fine les réorganisations spatiales de l'ADNr, nous avons développé une analyse d'image dédiée permettant de sonder l'organisation de cette fibre en trois dimensions. Nous avons révélé une structure sous-jacente de l'ADNr composée d'une succession de domaines organisés spatialement par les cohésines. Cette étude ouvre des perspectives vers une meilleure compréhension de la régulation des cohésines dans l'organisation du génome<br>Cohesin is an evolutionary-conserved complex composed of a ring capable of DNA entrapment and of auxiliary proteins regulating its association with DNA. On the one hand, cohesin confers sister chromatid cohesion required for their proper segregation and on the other hand it establishes and maintains chromatin looping. Chromatin loops are crucial for assembly of topological domains, gene expression and genome stability. However, mechanisms driving their establishment remain to be elucidated. According to loop extrusion model, cohesin would capture small loops and enlarge them by extruding DNA throughout its ring. This model predicts that loop size would depend on both cohesin residence time on DNA and on its processivity. Deciphering cohesin regulation is thus fundamental to understand chromosome biology. In this study, we showed that mitotic chromosome arms of yeast Saccharomyces cerevisiae are organised in cohesin-dependent chromatin loops. We studied the role of cohesin regulatory subunits Pds5, Wpl1 and Eco1 on loop establishment. Our data show that Pds5 inhibits loop expansion via Wpl1 and Eco1. As previously described in mammals, Wpl1 counteracts loop expansion by dissociating cohesin from DNA. Our results suggest that Eco1 would inhibit cohesin translocation on DNA, required for loop expansion. We then studied how these proteins contribute to the organisation of the ribosomal DNA array (rDNA), a cohesin-rich, highly transcribed sequence segregated away from the rest of the genome. Our data point toward a central role for Pds5 in organising this genomic region, independently of Wpl1 and Eco1. To study in detail rDNA spatial organisation, we developed a dedicated image analysis to assess its organisation in three dimensions. We have unveiled an underlying organisation for rDNA, made by a succession of small domains spatially organised by cohesin. This study opens large perspectives towards a better understanding of cohesin regulation in genome organisation

Thesis (Ph.D. in Molecular Biology) -- University of Colorado, 2006.<br>Typescript. Includes bibliographical references (leaves 126-147). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G1 phase assembly of pre-replicative complexes. Only the Orc1-5 subunits are required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. In comparison to other eukaryotic Orc6 proteins, budding yeast Orc6 appears to be quite divergent. Two-hybrid analysis revealed that Orc6 only weakly interacts with other ORC subunits. In this assay Orc6 showed a strong ability to self-associate, although the significance of this dimerization or multimerization remains unclear. Imaging of Orc6-eYFP revealed a punctate sub-nuclear localization pattern throughout the cell cycle, representing the first visualization of replication foci in live budding yeast cells. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations from metazoans. An essential role for Orc6 in DNA replication was identified by depleting the protein before and during G1 phase. Surprisingly, Orc6 was required for entry into S phase after pre-replicative complex formation, in contrast to what has been observed for other ORC subunits. When Orc6 was depleted in late G1, Mcm2 and Mcm10 were displaced from chromatin, the efficiency of replication origin firing was severely compromised, and cells failed to progress through S phase. Depletion of Orc6 late in the cell cycle indicated that it was not required for mitosis or cytokinesis. However, Orc6 was shown to be associated with proteins involved in regulating these processes, suggesting that it may act as a signal to mark the completion of DNA replication and allow mitosis to commence.

Casein kinase 2 (CK2) is a ubiquitous, essential and highly conserved eukaryotic kinase. It phosphorylates more than 300 substrates, but its physiological role and regulation mechanism are still poorly understood (Meggio and Pinna, 2003). CK2 is traditionally considered to be a tetrameric enzyme, composed of two catalytic subunits and two regulatory subunits, which are encoded in yeast by CKA1 and CKA2 genes, CKB1 and CKB2 genes respectively. Deletion of regulatory subunits, or of either catalytic subunit gene alone has few phenotypic effects, but simultaneous disruption of both CKA1 and CKA2 genes is lethal. In Saccharomyces cerevisiae a specialization of the two catalytic subunits (α e α’) was shown: α subunit is involved in cell polarity, while α’ subunit is linked to cell-cycle regulation and was shown to be fundamental both in G1 phase and in mitosis. In fact, at restrictive temperature cka1Δcka2ts mutants arrest cell cycle with a terminal phenotype constituted of 50% of unbudded G1 cells and 50% of cells arrested in metaphase and anaphase (Hanna et al., 1995). Among CK2 substrates, many cell-cycle proteins are known, both in mammalian cells and in yeast (p21, p27, Cdc2, Cdk1, Cdc37) (Meggio and Pinna, 2003). CK2 also phosphorylates two key regulators of the G1/S transition in yeast: Sic1, the Cdk1-Clb5/6 inhibitor, which is phosphorylated by CK2 on Ser201 (Coccetti et al., 2004; Coccetti et al., 2006), and Cdc34, the E2 enzyme required for the ubiquitination of many cell-cycle proteins (among which Sic1), which is phosphorylated by CK2 on Ser207, Ser216 e Ser282 (Pyerin et al., 2005; Barz et al., 2006; Sadowski et al., 2007). My PhD research activity was focused on the relationship between CK2 and these two relevant substrates (Sic1 and Cdc34), in order to understand CK2-mediated regulation of the G1/S transition in yeast. Previous analysis showed an increase of Sic1 level in cka1Δcka2ts strain at 37°C (Coccetti et al., 2006); thus we investigated whether this increase was responsible for the G1 arrest of this strain at restrictive temperature. We showed that the observed increase of Sic1 level was responsible for an inhibition of Cdk1-Clb5 kinasic activity. Moreover, SIC1 deletion, like the shutting-down of its expression, in a cka1Δcka2ts background, bypassed the G1 arrest at 37°C, leading to a single cell-cycle arrest, in which cells showed a post-synthetic DNA content. These data, published in 2007 (Tripodi et al., 2007), explained for the first time the molecular mechanism of the G1 block due to CK2 inactivation: Sic1 accumulation inhibits Cdk1-Clb5 complex, thus preventing the onset of DNA replication. We then worked on Cdc34, the E2 enzyme involved in Sic1 degradation. Literature data and our computational analysis revealed that Cdc34 protein present many consensus sites for CK2 phosphorylation. Mass spectrometry (MS) analysis on recombinant Cdc34 phosphorylated in vitro by CK2 showed phosphorylations on the following sites: S130, S167, S188, S195, S207, S282. In particular, among these, S130 and S167, within the catalytic domain of the protein, are highly conserved among Cdc34 homologues in various organisms, and were identified as phosphorylated sites in vivo in a CK2-dependent manner. Through a thiolester assay, we studied Cdc34 (wild-type and Cdc34S130AS167A) binding to ubiquitin in vitro, and we observed that lack of CK2-mediated phosphorylation on S130 and S167 strongly reduced the ubiquitin-charging ability of Cdc34. Subsequent in vivo analysis allowed us to investigate the physiological role of these phosphorylations. We observed that Cdc34S130AS167A overexpression is not able to complement the thermo-sensitive mutation cdc34-2 (Schwob et al., 1994), and determined a double arrest with both pre-replicative and post-replicative DNA content; the G1 block was characterized by Sic1 accumulation and was bypassed by SIC1 deletion. Yet, Cdc34S130AS167A expression to a level comparable to the endogenous protein led to a uniform G1 arrest at restrictive temperature, like the arrest observed in the control strain cdc34-2ts. Thus, these data, published in 2008 (Coccetti et al., 2008), showed that CK2 phosphorylation of the catalytic domain of Cdc34 was required for the function of the enzyme and for the in vivo ubiquitination of its substrates (among which Sic1). A part from the study of CK2 substrate of G1 phase, we investigated if CK2 was regulated by nutritional conditions, which are important for the modulation of the cell-cycle and especially of the G1/S transition. We used yeast strains expressing TAP-tagged CK2 subunits, and we showed that neither total levels of the four subunits, nor their subcellular localization (which is mainly nuclear both in glucose and in ethanol) were modulated by carbon source. Then we measured CK2 activity, with a traditional assay and with a new assay that we developed using recombinant Sic1 as CK2 substrate; we showed that CK2 activity was clearly lower in ethanol growing cells than in glucose growing ones. We further investigated whether this difference in CK2 activity was due to the different growth rate or to the different carbon metabolism of cells growing in glucose or ethanol. To this purpose, we used bioreactor technology, to separately consider growth rate effects and metabolism effects. This system showed that growth rate was the main factor responsible for the modulation of CK2 activity. We also showed, by using mutant strains bearing a deletion in one of the two genes encoding for the catalytic subunits, that both subunits (α e α’) were regulated by nutritional conditions; moreover, α subunit seems to have a higher activity than α’ subunit. Therefore we provided the first evidence of a regulation of CK2 activity in yeast.

Der eukaryotische Zellzyklus ist ein streng regulierter Prozess, für dessen zeitlichen Ablauf unter anderem oszillierende Genexpression notwendig ist. Die Regulation und die zeitliche Koordination des Zellzyklus sind nach wie vor fundamentale Fragen der Zellbiologie. Spezifische Ereignisse, wie DNA Replikation und Zellkernteilung, können vier Zellzyklusphasen zugeordnet werden, welche durch Cyclin-abhängige Kinasen, Cycline und deren Inhibitoren reguliert werden. Während in Saccharomyces cerevisiae Cyclin-abhängige Kinasen (Cdc28, Pho85) über den gesamten Zellzyklus zu Verfügung stehen, werden Cycline und ihre Inhibitoren nur in spezifischen Phasen exprimiert. In S. cerevisiae sind drei wichtige G1-Cycline (Cln1-Cln3) in die oszillierende Genexpression involviert. In dieser Arbeit wurde die zeitaufgelöste, transkriptomweite Genexpression im Wildtyp und in Cyclindeletionsmutanten gemessen. Um die Rolle der G1-Cycline für die Feinabstimmung des Zellzykluses zu verstehen, wurden Gene nach charakteristischen Expressionsprofilen geclustert, Expressionsmaxima detektiert, ein Transkriptionsfaktornetzwerk integriert und Zellzyklusphasendauern bestimmt. Um Unterschiede zwischen der Rolle der Cycline zu verstehen, wurden die Zellen zusätzlich Osmostress ausgesetzt. Des Weiteren wurde mit Hilfe von RNA-Fluorescence In Situ Hybridization (FISH) die Expression zweier Cycline (PCL1 und PCL9), die an Pho85 binden, auf Einzelzellniveau gemessen. Um die Expression in spezifischen Zellzyklusphasen zu quantifizieren, wurden einzelne Zellen mithilfe von Zellzyklusmarkern spezifischen Zellzyklusphasen zugeordnet. Nachdem die Expression unter normalen Wachstumsbedingungen gemessen wurde, wurde zusätzlich Osmostress angewandt. Durch die Kombination einer Einzelzellquantifizierung und einer transkriptomweiten Methode konnten spezifische Aufgaben der Cycline, Cln1, Cln2 und Cln3, erforscht werden. Zusätzlich konnten backup Mechanismen für die Zellzyklusregulation entschlüsselt werden.<br>The eukaryotic cell cycle is a highly ordered process. For its timing and progression, oscillating gene expression is crucial. The stability of cell cycle regulation and the exact timing is still a fundamental question in cell biology. Specific events, like DNA replication and nuclear division can be assigned to four distinct phases. These events are regulated by cyclin-dependent kinases, cyclins and their inhibitors. In Saccharomyces cerevisiae cyclin-dependent kinases (Cdc28, Pho85) are present throughout the cell cycle, while cyclins and their inhibitors are only expressed and active during specific phases. The G1 cyclins Cln1-3 are essential players to induce oscillating gene expression and are thereby involved in the fine-tuning of the cell cycle. To understand the role of the G1 cyclins for exact cell cycle timing and oscillating gene expression, time-resolved, transcriptome-wide gene expression in wild type and cyclin deletion mutants were measured. Characteristic expression profiles were clustered, precise peak times for each gene were estimated, a transcription factor network was integrated and cell cycle phase durations were defined. To further understand the role and differences of each cyclin osmostress was applied. Furthermore the expression of two cyclins (PCL1 and PCL9) corresponding to the cyclin-dependent kinase Pho85 was measured in single cells. Using RNA-Fluorescence In Situ Hybridization (FISH) and cell cycle progression markers, high and low expression phases and absolute numbers of mRNAs were obtained. Gene expression was quantified under normal and osmostressed growth conditions to understand the necessity of the cyclins for osmostress adaptation in different cell cycle phases. By the combination of a single cell and a transcriptome-wide approach distinct roles of G1 cyclins Cln1, Cln2 and Cln3 were deciphered and an insight in the backup mechanisms during cell cycle progression for normal and osmostressed growth conditions were proposed.

Dans la levure S. cerevisiae, la mitose nécessite le positionnement du fuseau mitotique le long de l’axe cellule mère-bourgeon (future cellule fille) afin d‘assurer une bonne ségrégation des chromosomes. Ce phénomène requiert le fonctionnement de deux mécanismes impliquant les protéines Kar9 et Dyn1. Durant la métaphase, Kar9 se positionne de manière asymétrique le long du fuseau mitotique, avec une accumulation notable sur les microtubules qui émanent de l’ancien « spindle pole body » (SPB; l’équivalent du centrosome dans les vertébrés), qui est normalement dirigé vers le bourgeon. Dans le cas d’un défaut d’alignement du fuseau mitotique, un mécanisme appelé « Spindle Position Checkpoint » (SPOC) inhibe la sortie de mitose et la cytokinèse, afin de permettre un réalignement correct du fuseau mitotique. La principale cible de ce checkpoint est une GTPase Tem1. Dans le cas d’alignement correct du fuseau mitotique, Tem1 active une voie de signalisation appelée le « Mitotic Exit Network » (MEN) qui permet de mener à la sortie de mitose et à la cytokinèse. Lors de la transition métaphase/anaphase Tem1 se positionne asymétriquement sur les SPBs jusqu’à se concentrer majoritairement sur l’ancien SPB. Des données récentes ont montré que des composants du MEN, Tem1 inclus, sont également impliqués dans la régulation de la localisation de la protéine Kar9 à l’SPB, et dans l’établissement d’une polarité correcte des SPBs durant la métaphase. En effet, Kar9 se positionne plus symétriquement dans le cas des mutants du MEN que dans le type sauvage, ce qui engendre des problèmes d’orientation du fuseau et de ségrégation des SPBs. Nous cherchons à élucider comment l’activité du MEN régule la localisation de Kar9 et l’orientation du fuseau mitotique en métaphase alors que les fonctions du MEN liées à la sortie de mitose restent bloquées jusqu’à la télophase. Nous avons émis l’hypothèse que les modifications post-traductionnelles de Tem1 pourraient jouer un rôle dans la régulation du MEN. Il a été montré que les résidus Y40 et Y45 sont phosphoryles in vivo. Afin de disséquer le rôle de ces résidus nous les avons mutés en phénylalanines. Ces mutations peuvent complémenter la létalité induite par la délétion de TEM1, suggérant que ce mutant conserve les fonctions essentielles de Tem1. Par ailleurs, la cinétique de progression du cycle cellulaire du mutant est la même que celle du type sauvage, signifiant que la perte de phosphorylation sur Tem1 ne semble pas agir sur la sortie de mitose. De plus, l’allèle mutant n’affecte pas la localisation aux SPBs de Tem1 ni celle de sa « GTPase-activating protein » Bub2/Bfa1 durant le cycle cellulaire. Bien que l’activité GTPasique de la protéine Tem1-Y40F,Y45F soit réduite in vitro, les mutations ne causent pas des défauts de SPOC in vivo et le mutant répond efficacement au mauvais alignement de fuseau mitotique en s’arrêtant en anaphase. Tous ces résultats nous suggèrent que la perte de phosphorylation de Tem1 n’affecte pas les fonctions de fin de mitose de cette GTPase. Par contre, nous avons découvert que la phosphorylation de Tem1 est requise pour la localisation asymétrique de Kar9 sur les SPBs, ainsi que pour l’alignement correct du fuseau mitotique durant la métaphase (la distribution de Kar9 est plus symétrique dans les cellules TEM1-Y40F,Y45F et que le fuseau mitotique n’est pas aligné correctement). Nous cherchons alors à trouver quelle kinase phosphoryle Tem1 et régule son activité. Les kinases potentielles sont la protéine Swe1 (la seule vraie kinase phosphorylant les tyrosines dans la levure) ainsi que la kinase Mps1 (kinase qui contrôle la duplication des SPBs). Nous développons actuellement des outils nous permettant de vérifier l’implication de ces deux candidats. Mots clés : Tem1, Kar9, cycle cellulaire, Mitotic Exit Network (MEN), Spindle Position Checkpoint (SPOC), phosphorylation on tyrosines<br>In the budding yeast Saccharomyces cerevisiae a faithful mitosis requires positioning of the mitotic spindle along the mother-bud axis to ensure proper chromosome segregation. This is achieved by two distinct but functionally redundant mechanisms that require the APC (adenomatous polyposis coli)-like protein Kar9 and dynein (Dyn1), respectively. During metaphase, Kar9 localizes asymmetrically on the mitotic spindle, with a prominent accumulation on astral microtubules emanating from the old spindle pole body (SPB – i.e. the yeast equivalent of the centrosome) that is normally directed towards the bud. In case of spindle misalignment, a surveillance mechanism called Spindle Position Checkpoint (SPOC) inhibits mitotic exit and cytokinesis, thereby providing the time necessary to correct spindle alignment. The main target of the SPOC is the small GTPase Tem1, which activates a signal transduction cascade called Mitotic Exit Network (MEN) that drives cells out of mitosis and triggers cytokinesis. Tem1 is localized at SPBs, with an increasingly asymmetric pattern during the progression from metaphase to anaphase, when Tem1 is concentrated on bud-directed old SPB. Recent data have implicated MEN components also in the regulation of Kar9 localization at SPBs and in setting the right polarity of SPBs inheritance during metaphase. In particular, Kar9 localizes more symmetrically in MEN mutants than in wild type cells and this leads to spindle orientation and SPB inheritance defects (i.e. with the new SPB being oriented towards the bud). A key question emerging from these data is how MEN activity is regulated to promote proper Kar9 localization and spindle positioning in metaphase, while being restrained until telophase for what concerns its mitotic exit and cytokinetic functions. We hypothesised that Tem1 post-translational modifications might be relevant for this control and for this reason we have been focusing on the role of Tem1 phosphorylation. Tem1 was found in a wide phosphoproteomic study to be phosphorylated on two tyrosines (Y40 and Y45) located at its N-terminus. We constructed a non-phosphorylatable mutant, TEM1-Y40F,Y45F, where the two phosphorylated tyrosines were mutated to phenylalanine. This mutant allele was able to rescue the lethality caused by TEM1 deletion, suggesting that it retains all its the essential functions. The kinetics of cell cycle progression of TEM1-Y40F,Y45F cells was similar to that of wild type cells, suggesting that lack of Tem1 phosphorylation is unlikely to affect mitotic exit. In addition, the TEM1-Y40F,Y45F allele did not affect the SPB localization of Tem1 and its regulatory GTPase-activating protein Bub2/Bfa1 during the cell cycle. Moreover, although the Tem1-Y40F,Y45F mutant protein showed reduced GTPase activity in vitro, it did not cause SPOC defects in vivo and could efficiently respond to spindle mispositioning. Altogether, these results suggest that lack of Tem1 phosphorylation does not affect the late mitotic functions of the GTPase. In contrast, we found that Tem1 phosphorylation is required for Kar9 asymmetry at SPBs and proper spindle positioning during metaphase. Indeed, TEM1-Y40F,Y45F cells display a more symmetric pattern of Kar9 distribution at SPBs in this cell cycle stage, as well as spindle position and orientation defects. We are currently investigating if Tem1 phosphorylation also regulates the pattern of SPB inheritance. Finally, an important question that we are trying to answer is “what is the kinase that phosphorylates Tem1?” The best candidates are the wee1-like kinase Swe1, which is the only true tyrosine kinase of budding yeast, and Mps1, a dual-specificity protein kinase controlling SPB duplication. While we are developing specific tools to study Tem1 phosphorylation and ultimately identify its promoting kinase, we gained preliminary data suggesting that both kinases might be involved in spindle positioning

Progression through the cell cycle is tightly controlled, and the decision whether or not to enter a new cell cycle can be influenced by both internal and external cues. For budding yeast one such external cue is pheromone treatment, which can induce G1 arrest. Two distinct mechanisms are known to be involved in this arrest, one dependent on the arrest protein Far1 and one independent of Far1, but the exact mechanisms have remained enigmatic. The studies presented here further elucidate both of these mechanisms. We looked at two distinct aspects of the Far1-independent arrest mechanism. First, we studied the role of the G1/S regulatory system in G1 arrest. We found that deletion of the G1/S transcriptional repressors Whi5 and Stb1 compromises Far1-independent arrest, but only partially, and that this partial arrest failure correlates to partial de-repression of G1/S transcripts. Deletion of the CKI Sic1, however, is more strongly required for arrest in the absence of Far1, though not when Far1 is present. Together, this demonstrates that functionally overlapping regulatory circuits controlling the G1/S transition collectively provide robustness to the G1 arrest response. We also sought to reexamine the phenomenon of pheromone-induced loss of G1/S cyclin proteins, which we suspected could be another Far1-independent arrest mechanism. We confirmed that pheromone treatment has an effect on G1 cyclin protein levels independent of transcriptional control. Our findings suggest that this phenomenon is dependent on SCFGrr1but is at least partly independent of Cdc28 activity, the CDK phosphorylation sites in Cln2, and Far1. We were not, however, able to obtain evidence that pheromone increases the degradation rate of Cln1/2, which raises the possibility that pheromone reduces their synthesis rate instead. Finally, we also studied the function of Far1 during pheromone-induced G1 arrest. Although it has been assumed that Far1 acts as a G1/S cyclin specific CDK inhibitor, there has been no conclusive evidence that this is the case. Our data, however, suggests that at least part of Far1’s function may actually be to interfere with Cln-CDK/substrate interactions since we saw a significant decrease of co-pulldown of Cln2 and substrates after treatment with pheromone. All together, the results presented here demonstrate that there are numerous independent mechanisms in place to help robustly arrest cells in G1.

Progression through the cell cycle is tightly controlled, and the decision whether or not to enter a new cell cycle can be influenced by both internal and external cues. For budding yeast one such external cue is pheromone treatment, which can induce G1 arrest. Two distinct mechanisms are known to be involved in this arrest, one dependent on the arrest protein Far1 and one independent of Far1, but the exact mechanisms have remained enigmatic. The studies presented here further elucidate both of these mechanisms. We looked at two distinct aspects of the Far1-independent arrest mechanism. First, we studied the role of the G1/S regulatory system in G1 arrest. We found that deletion of the G1/S transcriptional repressors Whi5 and Stb1 compromises Far1-independent arrest, but only partially, and that this partial arrest failure correlates to partial de-repression of G1/S transcripts. Deletion of the CKI Sic1, however, is more strongly required for arrest in the absence of Far1, though not when Far1 is present. Together, this demonstrates that functionally overlapping regulatory circuits controlling the G1/S transition collectively provide robustness to the G1 arrest response. We also sought to reexamine the phenomenon of pheromone-induced loss of G1/S cyclin proteins, which we suspected could be another Far1-independent arrest mechanism. We confirmed that pheromone treatment has an effect on G1 cyclin protein levels independent of transcriptional control. Our findings suggest that this phenomenon is dependent on SCFGrr1but is at least partly independent of Cdc28 activity, the CDK phosphorylation sites in Cln2, and Far1. We were not, however, able to obtain evidence that pheromone increases the degradation rate of Cln1/2, which raises the possibility that pheromone reduces their synthesis rate instead. Finally, we also studied the function of Far1 during pheromone-induced G1 arrest. Although it has been assumed that Far1 acts as a G1/S cyclin specific CDK inhibitor, there has been no conclusive evidence that this is the case. Our data, however, suggests that at least part of Far1’s function may actually be to interfere with Cln-CDK/substrate interactions since we saw a significant decrease of co-pulldown of Cln2 and substrates after treatment with pheromone. All together, the results presented here demonstrate that there are numerous independent mechanisms in place to help robustly arrest cells in G1.

Several cell cycle events require specific forms of the cyclin-CDK complexes. It has been known for some time that cyclins not only contribute by activating the CDK but also by choosing substrates and/or specifying the location of the CDK holoenzyme. There are several examples of B-type cyclins identifying certain peptide motifs in their specific substrates through a conserved region in their structure. Such interactions were not known for the G1 class of cyclins, which are instrumental in helping the cell decide whether or not to commit to a new cell cycle, a function that is non-redundant with B-type cylins in budding yeast. In this dissertation, I have presented evidence that some G1 cyclins in budding yeast, Cln1/2, specifically identify substrates by interacting with a leucine-proline rich sequence different from the ones used by B-type cyclins. These “LP” type docking motifs determine cyclin specificity, promote phosphorylation of suboptimal CDK sites and multi-site phosphorylation of substrates both in vivo and in vitro. Subsequently, we have discovered the substrate-binding region in Cln2 and further showed that this region is highly conserved amongst a variety of fungal G1 cyclins from budding yeasts to molds and mushrooms, thus suggesting a conserved function across fungal evolution. Interestingly, this region is close to but not same as the one implicated in B-type cyclins to binding substrates. We discovered that the main effect of obliterating this interaction is to delay cell cycle entry in budding yeast, such that cells begin DNA replication and budding only at a larger than normal cell size, possibly resulting from incomplete multi-site phosphorylation of several key substrates. The docking-deficient Cln2 was also defective in promoting polarized bud morphogenesis. Quite interestingly, we found that a CDK inhibitor, Far1, could regulate the Cln2-CDK1 activity partly by inhibiting the Cln2-substrate interaction, thus demonstrating that docking interactions can be targets of regulation. Finally, by studying many fungal cyclins exogenously expressed in budding yeast, we discovered that some have the ability to make the CDK hyper-potent, which suggests that these cyclins confer special properties to the CDK. My work provides mechanistic clues for cyclinspecific events during the cell cycle, demonstrates the usefulness of synthetic strategies in problem solving and also possibly resolves long-standing uncertainties regarding functions of some cell cycle proteins.

Orientador: Guilherme Targino Valente<br>Resumo: A intensa utilização de combustíveis fósseis gerapreocupações constantes devido aos impactos de sua combustão ao meio ambiente. Os biocombustíveis são uma alternativa viável aos combustíveis fósseis por apresentarem vantagens como serem menos agressivos ao meio ambiente. O bioetanol é um dos biocombustíveis mais utilizados no mundo e sua produção pode ser feita pela fermentação realizada pela levedura Saccharomyces cerevisiae. No entanto, altas concentrações de etanol inibem diversos mecanismos biológicos da levedura, causando a diminuição da produtividade. A partir de resultados prévios, observou-se que o ciclo celular é uma das vias mais afetadas pelo etanol e, além disso, constatou-se a presença de lncRNAs regulando esta via emduas linhagens de S. cerevisiae, a BY4742 e SEY6210. Utilizando operadores Booleanos, um modelo lógico discreto foi desenvolvido para o ciclo celular no qual os nós do sistema assumem até quatro valores discretos que representam a quantidade ou o graude ativaçãodesses nós. O modelo desenvolvido apresentou boa performance preditiva, acertando 87.27% dos 109 fenótipos obtidosda literatura, tornando possível a simulação de novos elementos. Experimentos prévios demonstraram que as leveduras de baixatolerância ao etanol conseguem retomar o crescimento mais rápido do que as de alta tolerância. Nesse trabalho, simulações feitas com dados de expressão diferencial via RNA-Seq permitiu inferir que isso ocorre porque as linhagens de baixa tolerância sofrem arre... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: The intense use of fossil fuels raised concern about the future due to their negative environmental impact. Bio-fuels are alternatives to the fossil fuels due to be biodegradable and less environmentally harmful. The bio-ethanol is one of the most popular bio-fuel. It can be produced by fermentation using the yeast Saccharomyces cereviae. However, high ethanol concentration inhibits the yeast decreasing the ethanol yield. Previous data of our groups showed the cell cycle is one of most affected pathways during ethanol stress. Moreover, it was found lncRNAs regulating this pathway in the BY4742 and SEY6210 strains. Using Boolean operators the discrete logical model of the cell cycle was developed. The nodes may get up to four discrete values to represent theirs abundance of activation degree. This model correctly modeled around 87.27% of correct predictions based on 109 phenotypes from the literature, hence, this model is desirable to predict cell cycle behavior after addition of new elements. According to previous data of our group, the lower tolerant strains recover the normal growth faster than higher tolerant strains after stress relief. The simulations here presented by adding RNASeq information into the model, showed a cell cycle arrest at final phase of the cell cycle (M phase) in lower tolerant strains whereas in the higher tolerant ones this arrest occurs at the first phase (G1 phase) during the ethanol treatment. The simulations also indicated that in SEY6210 (low to... (Complete abstract click electronic access below)<br>Mestre

Heterochromatin in the budding yeast Saccharomyces cerevisiae is composed of polymers of the SIR (Silent Information Regulator) complex bound to nucleosomal DNA. Assembly of heterochromatin requires all three proteins of the Sir complex: the histone deacetylase Sir2, and histone binding proteins Sir3 and Sir4. Heterochromatin establishment requires passage through at least one cell cycle, but is not dependent on replication. Inhibition of chromatin modifying enzymes may be a mechanism for how cells limit assembly. Dot1 dependent methylation of H3K79 is suggested to inhibit de novo assembly. Halving the levels of Sir4 in cells causes a loss of silencing, suggesting that Sir4 protein abundance regulates the assembly of heterochromatin. We examine de novo assembly using a single cell assay. Half the level of Sir4 affects establishment, but not the maintenance, of silencing at HM loci. Additional Sir4 accelerates the rate of assembly. Epistasis analysis suggests that Dot1 dependent chromatin modification may act upstream of Sir4 abundance. We hypothesize that dot1Δ mutants speed assembly by disrupting telomeric heterochromatin, which liberates Sir4 to act at the HM loci. Deletion of YKU70, which specifically disrupts telomeric silencing, also speeds de novo assembly, without altering the methylation of histone H3. Consistent with our model, we have shown that Sir4 abundance falls during pheromone and stationary phase arrests after which several cell cycles are required before silencing can be reestablished.

The nucleosome is the fundamental structural unit of DNA compaction in eukaryotic cells and is formed by the wrapping of 147 bp double stranded DNA around a histone octamer. Nucleosome organization plays a major role in controlling DNA accessibility to regulatory proteins, hence affecting cellular processes such as transcription, DNA replication and repair. Our study focuses on genome-wide nucleosome positioning in S. cerevisiae to explore nucleosome determinants and plasticity throughout the cell cycle and their interplay with gene expression based on cell mRNA abundance. We pursued the contribution of DNA physical properties on nucleosome organization around key regulatory regions such as TSSs and TTSs by analyzing genome-wide MNase-digestion profile of genomic DNA. We also implemented a systematic approach to standardize MNase-Seq experiments by minimizing the noise generated by extrinsic factors to enable an accurate analysis of the underlying principles of nucleosome positioning and dynamics. Moreover, we carried out a large-scale study of nucleosome plasticity throughout the cell cycle and its interplay with transcription based on a comparative analysis among nucleosome maps, gene expression data and MNase sensitivity assays. We then focused on nucleosome organization around DNA replication origins and its possible effect on origin activation. Finally, we sought to characterize centromeric nucleosome composition and its oscillation along cell cycle. During the course of these studies, we found that key regulatory regions such as 5’ and 3’ nucleosome free regions (NFRs) contain unusual physical properties that are intrinsic to genomic DNA. We further demonstrated that DNA physical properties and transcription factors act synergistically to define NFRs, especially in genes with an open promoter structure. Once NFR is defined, the nucleosome positioning around TSSs can be predicted by a simple statistical model, supporting the energy barrier model for nucleosome positioning determination. However, we also observed that nucleosomes are quite dynamic at distal 5’ NFRs and do have distinct regulatory mechanisms. Our comparative analysis of nucleosome organization along cell cycle revealed that chromatin exhibits a distinct configuration due to DNA replication-dependent organization at S phase, showing higher sensitivity to MNase and displaying fuzzier nucleosomes along the genome. Moreover, we observed different features at M phase, where chromatin compaction is the highest and displays a slightly different pattern than in G1 and G2 phases. Interestingly, these changes in chromatin organization are sudden and acute and only affect some regions of the genome, whereas the majority of genes present conserved nucleosome patterns along cell cycle. Our individual gene analysis disclosed that the largest changes take place in cell cycle-dependent genes, indicating the interplay between chromatin and transcription. Moreover, a distinct nucleosome organization at high and low transcription rates further supports this relationship. The detailed analysis around replication origins shows that they display slightly wider NFRs at G1 phase due to pre-Replication complex binding. Once the replication origins are active, nucleosomes partially occupy NFRs up to a certain extent due to constitutive binding of ORC. Moreover, we provided further evidence that early firing origins tend to have more ordered nucleosome organization than late firing origins. Finally we illustrated that centromeric nucleosomes display a perfect positioning, confirming their strong centromeric sequence-dependent recruitment to DNA. The characterization of histone composition under physiological cell conditions suggested that the octameric nucleosome assembly model is favored in centromeres. Yet, our analysis along cell cycle showed centromeric nucleosome dynamics, proposing that its composition might oscillate along cell cycle. Taken together, our accurate study provides a dynamic picture of nucleosome positioning and its determinants; new insights into cell cycle-dependent chromatin organization on key regulatory regions and its interplay with gene expression; and adds a new dimension to the characterization of centromeric nucleosomes.<br>Nuestro estudio se centra en el posicionamiento de nucleosomas a nivel genómico en levadura, con tal de explorar los factores determinantes de nucleosomas y su plasticidad a lo largo del ciclo celular, así como su relación con la expresión génica basándonos en la cantidad de mARN celular. Encontramos que las regiones libres de nucleosomas (NFRs en inglés) en 5’ y 3’ contienen propiedades físicas inusuales, las cuales son intrínsecas del ADN genómico. Además, demostramos que estas propiedades físicas actúan sinérgicamente con factores de transcripción para definir las NFRs. Una vez la NFR está definida, el posicionamiento de nucleosomas en torno al inicio de transcripción (TSS en inglés) puede predecirse con modelos estadísticos simples. No obstante, también observamos que los nucleosomas son bastante dinámicos en las regiones distales a 5’NFRs y poseen distintos mecanismos reguladores. Nuestro análisis comparativo acerca de la organización de los nucleosomas reveló que la cromatina de hecho exhibe una configuración distinta debido al reordenamiento dependiente de la replicación en fase S, mostrando una mayor sensibilidad de corte por el enzima MNase y un mayor número de nucleosomas deslocalizados a lo largo del genoma. Adicionalmente, observamos características particulares en fase M, donde la cromatina sufre un mayor grado de compactación. Notablemente, estos cambios en la organización de la cromatina son repentinos y agudos y sólo afectan a algunas regiones del genoma, mientras que la mayoría de genes presentan una conservación del patrón de nucleosomas a lo largo del ciclo celular. El análisis detallado en torno a los orígenes de replicación muestra una NFR más ancha en fase G1, debido a la unión del complejo pre-replicatorio. Una vez se activa el origen, los nucleosomas sólo ocupan parcialmente la NFR, debido a la unión constitutiva del complejo de origen de replicación (ORC en inglés). También proporcionamos evidencias de que los orígenes tempranos tienden a tener una organización nucleosomal más ordenada que los tardíos. Finalmente, ilustramos que los nucleosomas centroméricos poseen un posicionamiento idóneo y asimismo, un ensamblaje distinto. Sin embargo, nuestro análisis también mostró la dinámica de los nucleosomas centroméricos a lo largo del ciclo celular, indicando que de hecho su composición puede oscilar a lo largo del ciclo celular. En conjunto, nuestro detallado estudio proporciona una imagen dinámica del posicionamiento de nucleosomas y sus factores determinantes; nuevos indicios respecto a la organización de la cromatina en regiones reguladoras clave en base al ciclo celular y su conexión con la expresión génica; y finalmente, añade una nueva dimensión a la caracterización de los nucleosomas centroméricos.

The budding yeast Saccharomyces cerevisiae is a model organism for studies on cell cycle. For the survival of this cells a tight coordination of cell growth and division occurs at Start, a regulatory area of the cell cycle positioned immediately before beginning of S phase, at the G1-S boundary. Start is the event, or set of events, that commits a cell to a round of division. This mechanism is based on achieving of a critical cell size (protein content per cell at the onset of DNA replication, Ps) to enter into S phase. Ps increases in proportion with ploidy and is modulated by nutrients. In fact, in bach cultures, the average cell size remains at low levels during growth on non-fermentable substrates, while the average size of cells increases in a linear way with the specific growth rate only during growth on fermentable substrates. A genome-wide genetic analysis has suggested that cell size control could be due to ribosome biogenesis rate, one of the most energetic demanding processes in a cell and it is modulated according to nutrient availability. Indeed, a large cluster of genes involved in ribosome biogenesis, have been identified in a screen for small size (whi) mutants. This includes SFP1 and SCH9 genes. The first encode a zinc finger protein, promoting the transcription of a large cluster of genes involved in ribosome biogenesis, where the latter is serine threonine protein kinase involved in stress response and nutrient-sensing signaling pathway. Recent work from our laboratory allowed to identify that Far1, a cyclin kinase dependent inhibitor, and Cln3, a G1 phase cyclin, may form a nutritional modulated threshold controlling the entrance into S phase. Two parallel pathways downstream from the TORC1 complex regulate expression of genes encoding ribosomal proteins (RP) and the so-called RiBi regulon, composed by genes involved in ribosome biogenesis. The two pathways involve Sfp1, and Sch9. Therefore it was of interest to see whether the increase in size (RNA and protein) brought about by FAR1 overexpression was mediated by Sfp1 and Sch9. The effect of FAR1 overexpression on cell size parameters in the wild type BY4741 strain (isogenic to the sch9Δ and sfp1Δ mutants), grown in synthetic complete media supplemented with either ethanol or glucose as a carbon source, was similar to that reported in the W303 background. sfp1Δ and sch9Δ mutants were much smaller than wild type both in glucose - confirming previous data (Jorgensen et al., 2002; Jorgensen et al., 2004) - and ethanol-supplemented media. As observed in wild type cells, in both mutant strains FAR1 overexpression had only minor effects on cell cycle and cell size related parameters on glucose-grown cells. FAR1 overexpression did not affect duplication time in ethanol-grown sch9Δ cells, while sfp1Δ mutants overexpressing the FAR1 gene product were quite unhealthy with a large increase in duplication time. Overall increase in cell size was dramatic in ethanol-grown cells: however, while in wild type cells and sch9Δ mutants the increase in cell size derived from a balanced increase in RNA and protein content, in the sfp1Δ mutant the increase in protein content was not accompanied by an increase in RNA content, as shown by both FACS and chemical analysis, indicating that the Sfp1 is required to maintain proper coupling of RNA and protein syntheses when the Far1 protein is overexpressed in ethanol-grown-cells. The observation that FAR1 overexpression has different effects in sfp1Δ cells grown in ethanol and glucose media was not entirely unexpected. First, in untransformed cells, the Far1 level of glucose-grown cells is larger than in ethanol-grown cells, while ectopically expressed Far1 accumulates to a similar level regardless of the carbon source: as a result, Far1 overexpression is more dramatic in ethanol-grown cells than in glucose-grown cells (Alberghina et al., 2004). Accordingly, the effect of Far1 overexpression on cell size are minor on cells grown in glucose-supplemented media and much more dramatic in ethanol-grown cells. In the second part of this study we try to determine whether (and possibly, to which extent) the regulatory function of glucose can be separated from its nutrient function. To this aim, we characterized yeast strains in which glucose sensing is strongly reduced. An essential requisite for the survival of free living microorganism like the budding yeast Saccharomyces cerevisiae is the capacity to regulate growth and cell cycle progression according to the frequent changes in the nutrient status, so that proliferation is rapid when large supplies of nutrients are available and ceases when these becomes exhausted. Nutrients like glucose must therefore generate signals that are somehow received and elaborated by the complex machinery governing growth and cell cycle progression. Besides being the favorite carbon and energy source for S. cerevisiae, glucose can act as a signaling molecule (“hormone”) to regulate multiple aspects of yeast physiology: addition of glucose to quiescent or ethanol growing cells triggers a fast and massive reconfiguration of the transcriptional program, which enables the switch to fermentative metabolism and promotes an outstanding increase of the cell biosynthetic capacity. Yeast cells evolved several mechanisms for monitoring glucose level in their habitat: the cAMP-PKA pathways (with its two branches comprising Ras and the Gpr1-Gpa2 module), the Rgt2/Snf3-Rgt1 pathway and the main repression pathway involving the kinase Snf1. In order to investigate whether the glucose effect on cell size was due to its function as nutrient, that require metabolism of the sugar, or to sensing of extracellular glucose levels, yeast strains in which one or more of the glucose sensing pathway was impaired, due to gene deletion of glucose receptors (GPR1, SNF3, RGT2), were analyzed. These mutants show only a partial nutritional modulation of cell size and/or of duplication time. The gpa2Δ gpr1Δ strain does not show substantial changes in duplication time compared to its isogenic wild type grown in the same conditions, while its protein content is consistently lower. In the snf3Δ rgt2Δ and in the snf3Δ rgt2Δ gpa2Δ gpr1Δ strains a lower protein content is accompanied by an increase in duplication time, when compared to wild type strain. Furthermore, in all glucose sensing mutants the variation of protein content, as function of glucose levels, is less than the wild type. In the presence of ethanol, the kinetic parameters of mutants strain analyzed are comparable to wild type: there is only a strong increase in the duplication time, while there isn’t a further decrease in protein content compared to 0.05% glucose concentration. Data obtained show that the Gpa2-Gpr1 pathway specifically modulates Ps setting, while the Snf3-Rgt2 pathway plays an important role in Ps and growth rate setting of the cells. In conclusion, this work highlighted that the elements involved in the cell size determination are multiple and interconnected. A strong alteration in cell size and protein content could originate not only from alteration in the dosage of genes involved in the molecular mechanism of the threshold which controls Ps, but also from environmental conditions. Of particular relevance seems to be that glucose effect is largely acting as a signaling molecule, rather than as an energy source. Further studies are necessary in order to clarify the molecular mechanism that link the glucose sensing to the molecular machinery responsible of G1-S transition.

Résumé : Le nucléole est considéré comme étant une « usine » à produire des ribosomes. Cette production est la fonction la plus énergivore de la cellule. Elle met en jeu les trois ARN polymérases et représente 80% de l’activité de transcription au sein d’une cellule. Les trois quarts de cette activité de transcription correspondent à la synthèse des ARNr par l’ARN polymérase I (ARNPI). Ainsi mieux comprendre les mécanismes cellulaires se déroulant à l’intérieur de ce compartiment permettra le développement de nouveaux traitements contre le cancer. La synthèse d’ARNr par l’ARNPI est régulée à trois niveaux : l’initiation de la transcription, l’élongation et le nombre de gènes de l’ARNr en transcription. La plupart des travaux qui se sont intéressés à ces niveaux de régulation ont été réalisés avec des cellules en phase exponentielle de croissance. Au cours de mes travaux, je me suis attardé sur la régulation de la transcription par l’ARNPI au cours de la phase G1 du cycle cellulaire et au début de la phase S. Ainsi mes résultats ont montré que si la chromatine des gènes de l’ARNr est essentiellement dépourvue de nucléosomes, la régulation de l’ARNPI diffère dans des cellules en G1 et au début de la phase S. J’ai pu de ce fait observer qu’en G1, la transcription de l’ARNPI se concentre sur un nombre réduit de gènes en transcription. Dans des cellules arrêtées au début de la phase S avec de l’hydroxyurée, la transcription de l’ARNPI est perturbée par un défaut de maturation de l’ARNR. Fort de ces résultats sur la nature des gènes ribosomaux en phase G1, je me suis attardé à la réparation de ces gènes lors de cette phase. Alors que dans des cellules en phase exponentielle de croissance irradiées avec des UVC, la chromatine des gènes de l’ARNr se ferme ; je n’ai pas observé la formation de nucléosomes suite à l’irradiation de cellules synchronisée en G1. Mes résultats montrent également que la réparation est plus efficace. Parallèlement, j’ai exploré l’assemblage du complexe de réparation par excision de nucléotides. Toutefois, les résultats obtenus sont peu concluants.<br>Abstract : The nucleolus is thought to be a “factory” involve in the production of ribosomes. This production is the most energetically consuming process in the cell. The three RNA polymerases are involved and this represents 80% of the total transcription activity of the cell. Three quarters of this transcriptional activity correspond to the synthesis of rRNA by the RNA polymerase I (RNAPI). So a better understanding of the cellular mechanisms taking place in this compartment may help for the development of new drugs against cancer. The synthesis of rRNA by RNAPI is regulated at three levels: initiation of transcription, elongation and the number of rRNA genes in transcription. Most of the works that characterized those levels of regulation were done in exponentially growing cells. During my work, I studied the regulation of RNAPI during the G1 phase of the cell cycle and during the early S phase. So my results have shown that if the chromatin of the rRNA genes mostly depleted of nucleosomes, the regulation of the RNAPI differs in cells in G1 and early S phase. I could observe that in G1, RNAPI transcription concentrates on a reduced number of transcribed rRNA genes. In cells arrested in early S phase with hydroxyurea, RNAPI transcription is disrupted by a defect in rRNA processing. With this results on the nature of the ribosomal genes in G1, I started the analysis of the DNA repair of those genes during this phase of the cell cycle. In UVC irradiated exponentially growing cells, the rRNA genes are closing. But in cells synchronized in G1, I could not observe the deposition of nucleosomes after UVC irradiation. Moreover, my results show an increase repair of the locus. In parallel, I have explored the assembly of the complex of nucleotide excision repair. However, the results were not conclusive.

Thesis (Ph.D. in Biochemistry & Molecular Genetics) -- University of Colorado at Denver and Health Sciences Center, 2006.<br>Typescript. Includes bibliographical references (leaves 147-164). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Les mutations du gène suppresseur de tumeur p53 représentent l'anomalie moléculaire la plus fréquemment observée dans les cancers suggérant que l'inactivation de ce gène constitue une étape clé de la transformation maligne. Le gène p53 code pour un facteur de transcription capable, en réponse à des conditions génotoxiques, de réguler l'expression des gènes p21 et Bax respectivement impliquées dans l'arrêt du cycle cellulaire et l'activation de l'apoptose. Les données à la fois structurales et fonctionnelles suggèrent que la conséquence majeure des mutations du gène p53 dans les cancers est la perte de l'activité transcriptionnelle de p53. Cette thèse a eu pour objet de développer de nouvelles méthodes d'analyse fonctionnelle du gène p53 permettant d'étudier les conséquences biologiques des mutations observées dans les cancers. Ces méthodes, basées sur l'analyse de la fonction transcriptionnelle dans la levure saccharomyces cerevisiae nous ont permis (i) de mettre au point un système d'étude de la mutagénèse du gène p53 in vitro, (ii) de développer des outils diagnostiques pour détecter sélectivement les mutations somatiques et constitutionnelles inactivant la fonction transcriptionnelle de p53, et (iii) d'identifier un transcrit alternatif de p53 non fonctionnel exprimé dans les lymphocytes humains normaux. Enfin, grâce à la mise au point de deux nouveaux systèmes biologiques dans la levure, nous avons pu montrer que l'effet des mutations sur l'activité transcriptionnelle de p53 était hétérogène. En effet si toutes les protéines mutantes analysées sont incapables de transactiver le gène Bax, certaines conservent l'aptitude à transactiver le gène p21. L'ensemble des travaux réalisés indique que l'une des conséquences majeures des mutations de p53 est la perte de l'activité transcriptionnelle, et illustre l'intérêt de la levure saccharomyces cerevisiae pour l'étude fonctionnelle des gènes humains.

Tato práce se zabývá buněčným cyklem kvasinky Saccgaromyces cerevisiae. Oblastí našeho zájmu je přechod mezi G1 a S fází, kde je naším cílem identifikovat velikosti buňky v době počátku DNA replikace. Nejprve se věnujeme nedávno publikovanému matematickému modelu, který popisuje mechanismy vedoucí k S fázi. Práce poskytuje detailní popis tohoto modelu, stejně jako časový průběh některých důležitých proteinů či jejich sloučenin. Dále se zabýváme pravděpodobnostním modelem aktivace replikačních počátků DNA. Nově uvažujeme vliv šíření DNA replikace mezi sousedícími počátky a analyzujeme jeho důsledky. Poskytujeme také senzitivní analýzu kritické velikosti buňky vzhledem ke konstantám popisujícím dynamiku reakcí v modelu G1/S přechodu.

DNA molecule is complex, fragile and can suffer different damages. Specific DNA repair mechanisms were evolved to respond to these challenges, and to allow a faithful transmission of genetic information throughout generations. If the damaging conditions are extensive, a mechanism called DNA damage checkpoint takes care of arresting the progression of the cell division cycle to allow the cell to repair the damage before proceeding further. Genes involved in the DNA damage checkpoint are conserved throughout evolution and mutations in the human genes are known to produce severe illnesses - like Ataxia Telangiectasia - and genomic instability, which is usually considered as the onset of cancer: indeed checkpoint genes, like BRCA1, were found to be mutated in different types of cancers. The yeast Saccharomyces cerevisiae has been widely used to study the DNA damage checkpoint because, despite its evolutionary distance, the easiness in generating knockout and mutant strains has facilitated the understanding of the underlying mechanisms. In this yeast, as in humans, the signal that activates the checkpoint is represented by the ssDNA covered by RPA, to which many different checkpoint and repair factors are recruited. ssDNA signals are responsible for the activation of Mec1 (hATR), the apical kinase of the checkpoint pathway, but in humans two other factors are required for this signalling to occur: a ring-like heterotrimer - the PCNA-like complex - which is loaded onto DNA in response to damage and which recruits the second factor, TopBP1. Once active, Mec1 kinase phosphorylates a series of substrates, among which there is the Ddc1 subunit of the PCNA-like complex, and the Rad9 protein; phosphorylated Rad9 allows the recruitment of Rad53, the central kinase of the checkpoint whose Mec1-dependent activation contributes to cell survival after DNA damage and replication stress. To be phosphorylated by DNA-bound Mec1, the Rad9 protein must be recruited to chromatin: this process involves the binding of a Rad9 domain - the Tudor domain - to a methylated lysine on histone H3. Indeed, cells mutated in the conserved H3 lysine, in the Tudor domain or in the histone methyltransferase Dot1 are defective in Rad9 and Rad53 phosphorylation when DNA is damaged in the G1 phase of the cell cycle. Surprisingly, when these mutants receive a DNA damage in mitosis, they are still able to phosphorylate Rad9 and Rad53, suggesting the presence of a second pathway that, in M phase, provides an alternative way for Rad9 to be phosphorylated. In this thesis evidences regarding this alternative pathway for Rad9 recruitment and phosphorylation are provided. This pathway depends upon the C-terminal tail of Dpb11, the yeast homologue of human TopBP1, and on the Mec1-dependent phosphorylation of threonine 602 of the Ddc1 subunit of the PCNA-like complex. We show that Dpb11 itself is phosphorylated after DNA damage and that this phosphorylation is reduced in the presence of a non-phosphorylatable 602-residue on Ddc1, suggesting that in these conditions Dpb11 cannot be functionally recruited. Supporting this idea the two-hybrid interaction between Ddc1 and Dpb11 requires the presence of a functional Mec1 kinase. Although being capable of in vitro stimulation of Mec1 kinase activity, after UV irradiation in M phase, Dpb11 is not required for Mec1 to phosphorylate its binding partner Ddc2. On the other hand, we provide evidences that Dpb11 performs its Mec1 activation task during the response to global replication stress; indeed Dpb11 and the PCNA-like complex are independently required to obtain a proper phosphorylation of histone H2A - here used as a marker of Mec1 kinase activity - and a full Rad53 activation. Consistent with this observation ddc1Δdpb11-1 mutants are extremely sensitive to chronic exposition to hydroxyurea, a commonly used chemotherapeutic drug that generates replication stress by reducing the concentration of dNTPs in the cell. We also provide evidence that this lethality is not due to classical checkpoint functions like the stabilisation of stalled replication forks or the ability to delay entrance in M phase. We suggest also that other proteins known to be involved in checkpoint activation after hydroxyurea treatment are working in the pathway in which Dpb11 is involved.