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. / 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.

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Bevor sich eine Zelle teilt muss sie ihr gesamtes genetisches Material verdoppeln. Eukaryotische Genome werden von einer Vielzahl von Replikationsstartpunkten, den sogenannten Origins, aus repliziert, die über das gesamte Genome verteilt sind. In dieser Arbeit wird der zugrundeliegende molekulare Mechanismus quantitativ analysiert, der für die nahezu simultane Initiierung der Origins exakt ein Mal pro Zellzyklus verantwortlich ist. Basierend auf umfangreichen experimentellen Studien, wird zunächst ein molekulares regulatorisches Netzwerk rekonstruiert, welches das Binden von Molekülen an die Origins beschreibt, an denen sich schließlich komplette Replikationskomplexe (RKs) bilden. Die molekularen Reaktionen werden dann in ein Differentialgleichungssystem übersetzt. Um dieses mathematische Modell zu parametrisieren, werden gemessene Proteinkonzentrationen als Anfangswerte verwendet, während kinetische Parametersätze in einen Optimierungsverfahren erzeugt werden, in welchem die Dauer, in der sich eine Mindestanzahl von RKs gebildet hat, minimiert wird. Das Modell identifiziert einen Konflikt zwischen einer schnellen Initiierung der Origins und einer effizienten Verhinderung der DNA Rereplikation. Modellanalysen deuten darauf hin, dass eine zeitlich verzögerte Origininitiierung verursacht durch die multiple Phosphorylierung der Proteine Sic1 und Sld2 durch Cyclin-abhängige Kinasen, G1-Cdk bzw. S-Cdk, essentiell für die Lösung dieses Konfliktes ist. Insbesondere verschafft die Mehrfach-Phosphorylierung von Sld2 durch S-Cdk eine zeitliche Verzögerung, die robust gegenüber Veränderungen in der S-Cdk Aktivierungskinetik ist und außerdem eine nahezu simultane Aktivierung der Origins ermöglicht. Die berechnete Verteilung der Fertigstellungszeiten der RKs, oder die Verteilung der Originaktivierungszeiten, wird auch genutzt, um die Konsequenzen bestimmter Mutationen im Assemblierungsprozess auf das Kopieren des genetischen Materials in der S Phase des Zellzyklus zu simulieren. / Before a cell divides it has to duplicate its entire genetic material. Eukaryotic genomes are replicated from multiple replication origins across the genome. This work is focused on the quantitative analysis of the underlying molecular mechanism that allows these origins to initiate DNA replication almost simultaneously and exactly once per cell cycle. Based on a vast amount of experimental findings, a molecular regulatory network is constructed that describes the assembly of the molecules at the replication origins that finally form complete replication complexes. Using mass–action kinetics, the molecular reactions are translated into a system of differential equations. To parameterize the mathematical model, the initial protein concentrations are taken from experimental data, while kinetic parameter sets are determined using an optimization approach, in particular a minimization of the duration, in which a minimum number of replication complexes has formed. The model identifies a conflict between the rapid initiation of replication origins and the efficient inhibition of DNA rereplication. Analyses of the model suggest that a time delay before the initiation of DNA replication provided by the multiple phosphorylations of the proteins Sic1 and Sld2 by cyclin-dependent kinases in G1 and S phase, G1-Cdk and S-Cdk, respectively, may be essential to solve this conflict. In particular, multisite phosphorylation of Sld2 by S-Cdk creates a time delay that is robust to changes in the S-Cdk activation kinetics and additionally allows the near-simultaneous activation of multiple replication origins. The calculated distribution of the assembly times of replication complexes, that is also the distribution of origin activation times, is then used to simulate the consequences of certain mutations in the assembly process on the copying of the genetic material in S phase of the cell cycle.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

BACKGROUND: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. METHODS/PRINCIPAL FINDINGS: Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. CONCLUSION: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle. / Dissertation

La division cellulaire est influencée par les différents stimuli provenant de l’extérieur ou de l’intérieur de la cellule. Plusieurs réseaux enzymatiques élaborés au cours de l’évolution relayent l’information générée par ces signaux. Les modules MAP kinases sont extrêmement importants au sein de la cellule. Chez l’humain, 14 MAP kinases sont regroupées en sept voies distinctes intervenant dans le contrôle d’une myriade de processus cellulaires. ERK3/4 sont des homologues de ERK1/2 pour lesquelles on ne connaît que très peu de choses concernant leurs fonctions et régulation. Ces MAP kinases sont dites atypiques puisqu’elles ont des particularités structurales et des modes de régulation qui diffèrent des autres MAP kinases classiques. Ainsi, notre laboratoire a démontré que l’activité de ERK3 est régulée par le système ubiquitine-protéasome et qu’elle pourrait avoir un rôle à jouer dans le contrôle de la différenciation et la prolifération cellulaire. La première étude présentée décrit la régulation de ERK3 au cours du cycle cellulaire. Nous avons observé que ERK3 est hyperphosphorylée et s’accumule spécifiquement au cours de la mitose. Des analyses de spectrométrie de masse ont mené à l’identification de quatre sites de phosphorylation situés à l’extrémité du domaine C-terminal. Nous avons pu démontrer que la kinase mitotique CDK1/cycline B phosphoryle ces sites et que les phosphatases CDC14A et CDC14B les déphosphorylent. Finalement, nous démontrons que la phosphorylation mitotique de ERK3 a pour effet de la stabiliser. Au début de mes études doctorales, la kinase MK5 fut identifiée comme premier partenaire et substrat de ERK3. MK5 a très peu de fonctions connues. Des données dans la littérature suggèrent qu’elle peut moduler le cycle cellulaire dans certaines conditions. Par exemple, MK5 a récemment été identifié comme inducteur de la sénescence induite par l’oncogène Ras. Dans la deuxième étude, nous décrivons une nouvelle fonction de MK5 dans le contrôle du cycle cellulaire. Nous démontrons par des expériences de gain et perte de fonction que MK5 ralentit l’entrée en mitose suite à un arrêt de la réplication. Cette fonction est dépendante de l’activité enzymatique de MK5 qui régule indirectement l’activité de CDK1/cycline B. Finalement, nous avons identifié Cdc25A comme un nouveau substrat in vitro de MK5 dont la surexpression supprime l’effet de MK5 sur l’entrée en mitose. En conclusion, nos résultats décrivent un nouveau mécanisme de régulation de ERK3 au cours de la mitose, ainsi qu’une nouvelle fonction pour MK5 dans le contrôle de l’entrée en mitose en réponse à des stress de la réplication. Ces résultats démontrent pour la première fois l’implication de ces protéines au cours de la transition G2/M. Nos travaux établissent de nouvelles pistes d’études pour mieux comprendre les rôles encore peu définis des kinases ERK3/4-MK5. / The process of cell division is largely influenced by extracellular and intracellular cues. Many enzymatic pathways refined during evolution propagate the information generated by those cues. MAP kinase modules are extremely important within the cells. Human genome encodes 14 MAP kinases genes grouped into seven distinct pathways involved in the control of many cellular processes. ERK3/4 are kinases homologous to ERK1/2. Very little is known about their regulation and molecular functions. These MAP kinases are described as being atypical based on their unique structural characteristics and mode of regulation. Our laboratory was the first to demonstrate that the activity of ERK3 is mainly regulated by the ubiquitin-proteasome system in proliferating cells. In addition, several lines of evidence suggest a role for ERK3 in the control of cell differentiation and proliferation. The first study presented herein documents the regulation of ERK3 during the cell cycle. We observed that ERK3 is hyperphosphorylated and accumulated specifically during mitosis. Mass spectrometry analyses led to the identification of four phosphorylation sites located in the C-terminal domain. We demonstrate that mitotic kinase CDK1/cyclin B phosphorylates these sites which are dephosphorylated by Cdc14A and Cdc14B phosphatases. Finally, we show that mitotic phosphorylation of ERK3 controls its stability. At the beginning of my Ph.D. training, the kinase MK5 was the first identified binding partner and substrate of ERK3. MK5 is implicated in very few cellular functions. Data suggest that under certain conditions it modulates cell cycle progression. For example, MK5 was recently identified as a tumor suppressor gene essential for ras-induced senescence. In the second study of this thesis, we describe a novel function of MK5 in cell cycle progression. Gain and loss of function experiments demonstrate that MK5 delays G2/M transition following replicative stress. This function depends on its catalytic activity to indirectly regulates CDK1/cyclin B. Finally, we identified Cdc25A as a good in vitro substrate for MK5. Interestingly, Cdc25A expression inhibits MK5-induced delay of entry into mitosis. In conclusion, our results described a novel mechanism of regulation of ERK3 during mitosis and a novel function of MK5 in the control of G2/M transition after replicative stress. These data demonstrate for the first time the relation between these kinases and the G2/M transition. Our work should contribute to a better understanding of the roles of ERK3/4-MK5 kinases.

Transient transfection of mammalian cell lines is an effective approach for recombinant protein production, which can provide milligrams to grams of proteins in two weeks from cloning of the corresponding cDNA. Native glycosylated proteins prepared via this approach can be used for various purposes in molecular biology, immunology or pharmaceutical industry, i.e. initial phase of pre-clinical therapeutic protein research. One of the most used mammalian host cell lines is the human embryonic kidney cell line, that can be easily cultivated and chemically transfected. The amount of proteins produced by transiently transfected human embryonic kidney cells can be enhanced by a whole range of factors, i.e. co-expression or direct addition of acidic fibroblast growth factor to the culture medium, co-expression of cell cycle regulating proteins or anti-apoptotic proteins. Expression plasmid pTW5 was prepared and further modified by gene insertion of aFGF, cell cycle regulator p18, p21 or p27 (cyclin-dependent kinase inhibitors) or apoptosis inhibitor bcl-2 or bcl-x. These plasmids were then used for optimization of HEK293T cell line expression system. The impact of every single regulator and their combinations, including hitherto undescribed effect of combination of cell cycle regulator and anti-apoptotic...