Dissertations / Theses on the topic 'Cell cycle progression'

Tumorigenesis is the result of uncontrolled cell growth due to the deregulation of cell cycle checkpoints 1. Period 2 (Per2) is a tumor suppressor that oscillate in expression in a 24-hour cycle 2, 3. Here, we show that Per2 interacts with the tumor suppressor protein p53. Both G1 and G2 checkpoint pathways involve a p53 dependent pathway which can trigger the cell to go through cell arrest or programmed cell death4. Understanding all the mitigating factors involved in regulating cell cycle progression under DNA damage can offer a better idea in how cells become immortal. Initially discovered through screening of a human liver cDNA library, the novel interaction between p53-Per2 was further documented using co-precipitation. Interestingly, under genotoxic stress conditions, p53 and Per2 were not found to bind which leads us to suspect that Per2 does not affect active p53 which may possibly be due to post translational modifications of its active state. Furthermore we investigated p53's ability to act as a transcription factor in the presence of Per2, showing that the Per2-p53 complex prevents p53 from binding to DNA. This implies that the tetramerization of p53 may also be another factor in Per2's ability to bind to p53. A truncated p53 lacking the last 30 amino acids that theoretically increase p53's ability to form a tetramer showed a drastic reduction in binding to Per2 5, 6. On the other hand, p53 lacking the tetramerization domain showed binding similar to wildtype. Consequently we speculate that the ability of Per2 to modulate p53 and act as a tumor suppressor protein may be dependent on either the post translational modifications of p53 or its oligomeric state.
Master of Science

mTOR (Mammalian Target ofRapamycin), PI3K (Phosphatidylinositol3-kinase) and MEK (Mitogen-activated protein kinase/ERK kinase) have been shown to be potent regulators ofS6Kl at G₁ phase of the cell cycle. Research has been concentrated at the Gt phase to elucidate mTOR's role in cell growth and proliferation. Limited information is available on the activity ofmTOR, PI3K and ERKl/2 in cell cycle phases other than G₁. Since we have observed that S6Kl is active in phases other than G₁ our goal was to ascertain ifmTOR, PI3K or ERKl/2 have a role in regulating S6Kl during these cell cycle phases. Using cell cycle analysis and immunoblot analysis we have determined here that mTORand PI3K could play a role in regulating S6Kl at the G₁/S transition iQ. the cell cycle but there is also indications that mTOR and PI3K are potentially involved in regulating S6Kl in the phases post-G₁/S of the cell cycle, indicating a complex interaction between the kinases used to regulate S6Kl during the cell cycle. ERKl/2 is demonstrated to have limited involvement in the regulation of S6Kl during the cell cycle.

ATRX is a chromatin remodelling protein of the SNF2 family of chromatin remodelling proteins. Mutations in the ATRX gene have been shown to cause the ATR-X syndrome, an X-linked mental retardation disorder. ATRX is part of a chromatin-remodelling complex with Daxx that localizes to PML nuclear bodies or pericentromeric heterochromatin and is thought to regulate gene expression. In mice, Atrx inactivation results in embryonic lethality whereas conditional forebrain specific Atrx ablation showed impaired development and disorganization of the cortex. Furthermore, ATRX phosphorylation was shown to be cell cycle dependant, suggesting an important role for ATRX in cell cycle regulation. In this study we investigated the effects of ATRX downregulation in cell culture models, using siRNA transient transfection, a clone expressing an shRNA targeted to ATRX, and Atrxnull MEFs. ATRX downregulated cells showed reduced growth rates and cell cycle defects at the G1 and S phases of the cell cycle. Moreover, ATRX ablation was associated with an altered Rb phosphorylation status and decreased expression of the cyclin A and E2F-1 proteins. Taken together our results suggest that ATRX may play a significant role in cell cycle progression that is pertinent for proper development.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2006" Includes bibliographical references.

Selbstreproduktion ist eines der Kennzeichen aller lebenden Organismen. Der Zellzyklus dient der Selbstreproduktion in einzelligen Organismen. In mehrzelligen Organismen ist der Zellzyklus darüber hinaus für andere lebenswichtige Prozesse, einschließlich Immunreaktionen, unerlässlich. In dieser Arbeit wird eine Methode entwickelt mit der die Dauer der Zellzyklus Phasen bestimmt werden kann. Kenntnis über die Zellzyklusphasendauer ermöglicht vorherzusagen, wie schnell eine Population von proliferierenden Zellen wachsen wird, oder wie viele neue Zellen pro Stunde in einem Gewebe geboren werden. Im Kapitel 1 dieser Arbeit wird ein Zellzyklusmodell aufgestellt und mit experimentellen Bromdesoxyuridin Daten verglichen. Die Analyse zeigt, dass das Modell gut die experimentelle Kinetik beschreibt, hebt jedoch auch hervor dass einige der Parameter nicht identifiziert werden können. Dieses Problem wird in Kapitel 2 bearbeitet, wo zwei Ansätze erforscht werden, um den Informationsgehalt der Experimente zu erhöhen. In einem ersten Ansatz wird die Theorie der Versuchsplanung angewendet, um optimale Versuchspläne zu bestimmen. In einem zweiten Ansatz wird das übliche Bromdesoxyuridin Protokoll durch ein zweites Nukleosid erweitert. Beide Methoden verbessern in silico erheblich die Genauigkeit und Präzision der Abschätzungen. Im dritten Kapitel wird die Methodik in der Analyse der Keimzentrumsreaktion angewendet. Ein erheblicher Zufluss von Zellen in die dunkle Zone von Keimzentren wird vorhergesagt, und die Ansicht einer extrem schellen Zellteilung im Keimzentrum erscheint in dem Modell als ein Artefakt der Zellmigration.
Self-reproduction is one of the distinguishing marks of living organisms. The cell cycle is the underlying process by which self-reproduction is accomplished in single-celled organisms. In multi-cellular organisms, the cell cycle is in addition indispensable for other vital processes, including immune reactions. In this thesis a method is developed that allows to estimate the time it takes for a dividing cells to complete the CC phases. Knowledge of the CC phase durations allows to predict, for example, how fast a population of proliferating cells will grow in size, or how many new cells are born per hour in a given tissue. In Chapter 1 of this thesis, a cell cycle model with delays and variability in the completion times of each phase is developed. Analytical solutions are derived to describe a common experimental technique used for cell cycle analysis, namely pulse labeling with bromodeoxyuridine (BrdU). Comparison with data shows that the model reproduces closely measured cell cycle kinetics, however also reveals that some of the parameter values cannot be identified. This problem is addressed in Chapter 2. In a first approach, the framework of D-optimal experimental designs is employed, in order to choose optimal sampling schemes. In a second approach, the prevailing protocol with a single nucleoside is modified by adding a second nucleoside analog pulse. Both methods are tested and the results suggest that experimental design can significantly improve parameter estimation. In Chapter 3, the model is applied to the germinal center reaction. A substantial influx of cells into the dark zone of germinal centers is predicted. Moreover the wide-held view of rapid proliferation in germinal centers, appears, under this model, as an artifact of cell migration.

Through the years, several studies reported the involvement of nuclear lipid signalling as highly connected with cell cycle progression. Indeed, nuclear Phosphatidylinositol-4,5-Biphosphate (PIP2) hydrolisis mediated by Phospholipases C (PLC), which leads to production of the second messengers Diacylglycerol (DAG) and Inositol-1,4,5-Triphosphate (IP3), is a fundamental event for both G1/S and G2/M checkpoints. In particular, we found that nuclear DAG production was mediated by PLCbeta1, enzyme mainly localized in the nucleus of K562 human erythroleukemia cells. This event triggered the activation and nuclear translocation of PKCalpha, which, in turn, resulted able to affect cell cycle via modulation of Cyclin D3 and Cyclin B1, two important enzymes for G1/S transition and G2/M progression respectively.

Through the years, several studies reported the involvement of nuclear lipid signalling as highly connected with cell cycle progression. Indeed, nuclear Phosphatidylinositol-4,5-Biphosphate (PIP2) hydrolisis mediated by Phospholipases C (PLC), which leads to production of the second messengers Diacylglycerol (DAG) and Inositol-1,4,5-Triphosphate (IP3), is a fundamental event for both G1/S and G2/M checkpoints. In particular, we found that nuclear DAG production was mediated by PLCbeta1, enzyme mainly localized in the nucleus of K562 human erythroleukemia cells. This event triggered the activation and nuclear translocation of PKCalpha, which, in turn, resulted able to affect cell cycle via modulation of Cyclin D3 and Cyclin B1, two important enzymes for G1/S transition and G2/M progression respectively.

Cancer of the mammary gland is the most common type of cancer in women worldwide, and the vast majority of breast cancers originate from a cluster of malignant cells in the epithelial tissue of the breast, which initially confines the ductal carcinoma in situ. Research has shown that the signalling pathways that increase differentiation and maintain proliferation in normal epithelial cells are of utmost importance for sustaining this barrier against malignant cells. As a model for normal mammary epithelial cells, the MCF-12A cell line was used to determine factors that are required for cell cycle progression of these cells. A discontinuous treatment assay was developed in which the MCF-12A cells were treated with epidermal growth factor (EGF) and insulin at two distinct times to induce cell cycle re-entry. The use of these chemically defined growth factors enabled us to determine that continuous stimulation with mitogenic factors is not required for these cells to re-enter the cell cycle. An initial activation of the MAP kinase pathway and an up-regulation of the transcription factor c-Myc, followed by activation of the PI3K pathway, resulted in full competence to progress into S phase. The order in which the growth factors were applied, and thus the sequence in which the subsequent proteins were triggered, was of great importance for successful S phase entry. We found that estradiol (E2) was unable to induce the factors necessary for cell cycle progression. Furthermore, we report for the first time that E2 did not affect estrogen-regulated genes which normally are under the control of a ligand-bound estrogen receptor (ER). We suggest that the mechanism by which the ligand-activated ER usually interferes with the estrogen responsive element in the promoter region of the target genes is defective in the MCF-12A cell line. The results presented here may contribute to new approaches in chemotherapy, taking advantage of the diverse molecular mechanism in place for cell cycle progression and proliferation in malignant cells compared to normal mammary epithelial cells.

The work presented here describes novel functions for centrosome proteins, specifically for pericentrin and centriolin. The first chapter describes the involvement of pericentrin in ciliogenesis. Cells with reduced pericentrin levels were unable to form primary cilia in response to serum starvation. In addition we showed novel interactions between pericentrin, intraflagellar transport (IFT) proteins and polycystin 2 (PC2). Pericentrin was co-localized with IFT proteins and PC2 to the base of primary cilia and motile cilia. Ciliary function defects have been shown to be involved in many human diseases and IFT proteins and PC2 have been implicated in these diseases. We conclude that pericentrin is required for assembly of primary cilia possibly as an anchor for other proteins involved in primary cilia assembly. The second chapter describes identification of centriolin, a novel centriolar protein that localizes to subdistal appendages and is involved in cytokinesis and cell cycle progression. Depletion of centriolin leads to defects in the final stages of cytokinesis, where cells remain connected by thin intercellular bridges and are unable to complete abscission. The cytokinesis defects seemed to precede the G0/G1 p53 dependant cell cycle arrest. Finally, the third chapter is a continuation of the cytokinesis study and it identifies pericentrin as an interacting partner for centriolin. Like centriolin, pericentrin knockdown induces defects in the final stages of cytokinesis and leads to G0/G1 arrest. Moreover, pericentrin and centriolin interact biochemically and show codependency in their centrosome localization. We conclude that pericentrin and centriolin are members of the same pathway and are necessary for the final stages of cytokinesis.

Der Cholesterin-Stoffwechsel ist sehr streng auf zellulärer Ebene reguliert und ist essentiell für das Zellwachstum. MicroRNAs (miRNAs), eine Klasse nicht-kodierender RNAs, wurden als kritische Regulatoren der Genexpression identifiziert und entfalten ihre Wirkung vorwiegend auf posttranskriptioneller Ebene. Aktuelle Arbeiten aus der Gruppe um Fernández-Hernando haben gezeigt, dass hsa-miR-33a und hsa-miR-33b, miRNAs die in den Intronsequenzen der Gene für die Sterol-regulatorischen Element- Bindungsproteine (SREBP-2 und SREBP -1) lokalisiert sind, den Cholesterin-Stoffwechsel im Einklang mit ihren Wirtsgenen regulieren. Gleichermaßen inhibiert miR-33 Schlüsselenzyme in der Regulation der Fettsäureoxidation, einschließlich CROT, CPT1A, HADHB, SIRT6, AMPKα, genauso wie IRS2, eine wesentliche Komponente des Insulin-Signalwegs in der Leber. Diese Studie zeigt, dass hsa-miR-33 Familienmitglieder nicht nur Gene in Cholesterin- und Fettsäure-Stoffwechsel sowie Insulin-Signalwege regulieren, sondern zusätzlich die Expression von Genen des Zellzyklus und der Zellproliferation modulieren. miR-33 inhibiert die Expression der CDK6 und CCND1, wodurch sowohol die Zellproliferation als auch die Zellzyklusprogression verringert wird. Die Überexpression von miR-33 induziert einen signifikanten G1 Zellzyklusarrest. Durch eine Inhibierung der miR-33 Expression mittels 2''F/MOE-modifiziert Phosphorothioat-Backbone Antisense-Oligonukleotiden, wird die Leberregeneration nach partieller Hepatektomie (PH) in Mäusen verbessert, was auf eine wichtige Rolle für miR-33 in der Regulation der Hepatozytenproliferation während der Leberregeneration hinweist. Zusammengefasst zeigen diese Daten, dass Srebf/miR-33 Locus kooperieren, um Zellproliferation und Zellzyklusprogression zu regulieren, und könnte somit auch relevant für die menschliche Leberregeneration sein.
Cholesterol metabolism is tightly regulated at the cellular level and is essential for cellular growth. Cellular imbalances of cholesterol and fatty acid metabolism lead to pathological processes, including atherosclerosis and metabolic syndrome. MicroRNAs (miRNAs), a class of noncoding RNAs, have emerged as critical regulators of gene expression acting predominantly at posttranscriptional level. Recent work from Fernández-Hernando´s group and others has shown that hsa-miR-33a and hsa-miR-33b, miRNAs located within intronic sequences of the sterol regulatory element-binding protein (SREBP-2 and SREBP-1) genes, respectively, regulate cholesterol metabolism in concert with their host genes. Similarly, miR-33 targets key enzymes involved in the regulation of fatty acid oxidation including CROT, CPT1A, HADHB, SIRT6 and AMPKα, likewise, IRS2, an essential component of the insulin- signaling pathway in the liver. This study shows that hsa-miR-33 family members not only regulate genes involved in cholesterol and fatty acid metabolism and insulin signaling, but in addition modulate the expression of genes involved in cell cycle regulation and cell proliferation. Thus, miR-33 inhibited the expression of CDK6 and CCND1, thereby reducing cell proliferation and cell cycle progression. Over-expression of miR-33 induced a significant G1 cell cycle arrest and most importantly, inhibition of miR-33 expression using 2’F/MOE-modified phosphorothioate backbone antisense oligonucleotides improved liver regeneration after partial hepatectomy (PH) in mice, suggesting an important role for miR-33 in regulating hepatocyte proliferation during liver regeneration. Altogether, these data establish that Srebf/miR-33 locus may co-operate to regulate cell proliferation, cell cycle progression and may also be relevant to human liver regeneration.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.
Cataloged from PDF version of thesis.
Includes bibliographical references.
In budding yeast, aneuploidy has a detrimental effect in cell growth and proliferation. The work presented here shows that most aneuploid yeast strains delay cell cycle entry by increasing the critical size for budding and by decreasing the rate of volume accumulation during the G 1 phase of the cell cycle. This increase in the critical size for budding is due to in a delay in CLN2 mRNA accumulation and can be suppressed by supplying cells with high levels of this cyclin. Deletion of the cell cycle entry inhibitor WHI5 only partially suppressed the GI delay of aneuploid cells. These two results combined point to the possibility that aneuploidy might be interfering through a parallel pathway with the activation of the transcription factors Swi4 and Swi6. The growth defect seen in aneuploid cells is not due to gross defects in the translational machinery or lack of nutrients. Instead, yeast cells respond to aneuploidy by altering the translational efficiency of a number of genes. The results presented here indicate that aneuploidy affects entry into the cell cycle in at least two ways. The condition elicits a growth defect during the G 1 phase of the cell cycle and increases the critical size for budding.
by Christian Gonzalez Rubio.
Ph.D.

A subset of eukaryotic heterochromatin is located around the nucleoli, and this localization is correlated with gene silencing. Although there is some evidence for trans-acting factors organizing genomic loci around the nucleolus, the characterization of proteins and /or RNAs involved in perinculeolar heterochromatin localization and maintenance is incomplete. Notably, the mammalian female inactive X chromosome, a well-studied model of facultative heterochromatin, frequently resides in the perinucleolar regions during mid to late S phase. The disruption of the Xi–nucleolus association results in the erosion of heterochromatin compartment and silencing, which renders it a good model to investigate the mechanism and biological relevance of heterochromatin organization around the nucleolus. This dissertation will present evidence showing that Ki-67 regulates inactive X (Xi) chromosome association with nucleoli, maintains Xi heterochromatic structures, and regulates cell cycle progression, in cell-type-specific manner dependent on checkpoint proficiency. Ki-67 protein plays roles in heterochromatin organization during interphase. Upon Ki-67 depletion, a subset of Xi in human female hTERT-RPE1 moved away from nucleolus and displayed several features of compromised heterochromatin maintenance. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in virally transformed 293T cells upon Ki-67 depletion. Furthermore, I demonstrated that the different Xi heterochromatin alteration responses result from cell-type-specific reduced proportion of cells in S phase upon Ki-67 depletion. In human hTERT-RPE1, WI-38, IMR90, hTERT-BJ cell lines, depletion of Ki-67 slowed entry into S phase and coordinately downregulated genes related to DNA replication. These cell lines are able to induce p21 expression upon Ki-67 depletion. On the contrary, alteration of transcription and cell cycle progression were not observed in tumor-derived HeLa, U2OS and 293T cell lines. These cell lines do not induce p21 expression either. I additionally examined the Ki-67 function in mouse cell cultures. Depletion of Ki-67 neither redistributes inactive X chromosome nor regulates S phase progression in primary female mouse embryonic cells.

A subset of eukaryotic heterochromatin is located around the nucleoli, and this localization is correlated with gene silencing. Although there is some evidence for trans-acting factors organizing genomic loci around the nucleolus, the characterization of proteins and /or RNAs involved in perinculeolar heterochromatin localization and maintenance is incomplete. Notably, the mammalian female inactive X chromosome, a well-studied model of facultative heterochromatin, frequently resides in the perinucleolar regions during mid to late S phase. The disruption of the Xi–nucleolus association results in the erosion of heterochromatin compartment and silencing, which renders it a good model to investigate the mechanism and biological relevance of heterochromatin organization around the nucleolus. This dissertation will present evidence showing that Ki-67 regulates inactive X (Xi) chromosome association with nucleoli, maintains Xi heterochromatic structures, and regulates cell cycle progression, in cell-type-specific manner dependent on checkpoint proficiency. Ki-67 protein plays roles in heterochromatin organization during interphase. Upon Ki-67 depletion, a subset of Xi in human female hTERT-RPE1 moved away from nucleolus and displayed several features of compromised heterochromatin maintenance. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in virally transformed 293T cells upon Ki-67 depletion. Furthermore, I demonstrated that the different Xi heterochromatin alteration responses result from cell-type-specific reduced proportion of cells in S phase upon Ki-67 depletion. In human hTERT-RPE1, WI-38, IMR90, hTERT-BJ cell lines, depletion of Ki-67 slowed entry into S phase and coordinately downregulated genes related to DNA replication. These cell lines are able to induce p21 expression upon Ki-67 depletion. On the contrary, alteration of transcription and cell cycle progression were not observed in tumor-derived HeLa, U2OS and 293T cell lines. These cell lines do not induce p21 expression either. I additionally examined the Ki-67 function in mouse cell cultures. Depletion of Ki-67 neither redistributes inactive X chromosome nor regulates S phase progression in primary female mouse embryonic cells.

Differentiation and cell cycle regulation in stem cell have a key function for embryonic development, organ homeostasis and tissue repair. Recent results have shown that these two mechanisms are intrinsically connected. Indeed, cell cycle machinery directly controls maintenance of pluripotency and initiation of differentiation. More precisely, the cell cycle regulator Cyclin D appears to control the transcriptional activity of Activin/Nodal signalling during progression of the cell cycle in human Embryonic Stem Cells (hESCs). As a consequence, hESCs can only differentiate into endoderm in the Early G1 phase when Cyclin Ds are expressed at low levels. These results show the mechanisms by which the cell cycle defines differentiation propensity of stem cells. However, these observations also imply the existence of interplays coordinating extra cellular signalling pathways with the epigenetic state, chromatin structure and transcriptional networks during cell cycle progression and these mechanisms remain to be fully uncovered. Here, I have utilised the FUCCI reporter system combined with ATAC-Seq to analyse chromatin dynamics during cell cycle progression in hESCs. Furthermore, I performed ChIP-Seq analyses to define the genomic location of transcriptional regulators during cell cycle progression as well as RNA-Seq to confirm variation in gene expression pattern. Integration of these data shows that the chromatin status in hESCs is highly dynamic and the core pluripotency transcription factors and epigenetic modifiers change genomic location during cell cycle progression. I also showed that hESCs in the Late G1 phase accumulate transcripts that are important for differentiation and development; therefore, indicating this phase represents a unique portion of the cell cycle for cell fate decisions. Taken together, these results uncover that transcriptional networks are unexpectedly dynamic during the progression of cell cycle in stem cells. I hypothesise that these modifications are necessary to prime hESCs for different cell fate choices allowing a diversity of differentiation that is otherwise impossible. Overall these mechanisms underline the need to study transcriptional and epigenetic mechanisms in the dynamic context of the cell cycle and have major implications for adult tissue homeostasis and disease.

The eukaryotic cell cycle orchestrates the precise duplication and distribution of the genetic material, cytoplasm and membranes to daughter cells. In multicellular eukaryotes, cell cycle regulation also governs various organisatorial processes ranging from gametogenesis over multicellular development to tissue formation and repair. Consequently, defects in cell cycle regulation provoke a variety of human cancers. A global view of genes and pathways governing the human cell cycle would advance many research areas and may also deliver novel cancer targets. Therefore this work aimed on the genome-wide identification and systematic characterisation of genes required for cell cycle progression in human cells. I developed a highly specific and efficient RNA interference (RNAi) technology to realize the potential of RNAi for genome-wide screening of the genes essential for cell cycle progression in human tissue culture cells. This approach is based on the large-scale enzymatic digestion of long dsRNAs for the rapid and cost-efficient generation of libraries of highly complex pools of endoribonuclease-prepared siRNAs (esiRNAs). The analysis of the silencing efficiency and specificity of esiRNAs and siRNAs revealed that esiRNAs are as efficient for mRNA degradation as chemically synthesized siRNA designed with state-of-the-art design algorithms, while exhibiting a markedly reduced number of off-target effects. After demonstrating the effectiveness of this approach in a proof-of-concept study, I screened a genome-wide esiRNA library and used three assays to generate a quantitative and reproducible multi-parameter profile for the 1389 identified genes. The resulting phenotypic signatures were used to assign novel cell cycle functions to genes by combining hierarchical clustering, bioinformatics and proteomic data mining. This global perspective on gene functions in the human cell cycle presents a framework for the systematic documentation necessary for the understanding of cell cycle progression and its misregulation in diseases. The identification of novel genes with a role in human cell cycle progression is a starting point for an in-depth analysis of their specific functions, which requires the validation of the observed RNAi phenotype by genetic rescue, the study of the subcellular localisation and the identification of interaction partners of the expressed protein. One strategy to achieve these experimental goals is the expression of RNAi resistant and/or tagged transgenes. A major obstacle for transgenesis in mammalian tissue culture cells is the lack of efficient homologous recombination limiting the use of cultured mammalian cells as a real genetic system like yeast. I developed a technology circumventing this problem by expressing an orthologous gene from a closely related species including its regulatory sequences carried on a bacterial artificial chromosome (BAC). This technology allows physiological expression of the transgene, which cannot be achieved with conventional cDNA expression constructs. The use of the orthologous gene from a closely related species confers RNAi resistance to the transgene allowing the depletion of the endogenous gene by RNAi. Thus, this technology mimics homologous recombination by replacing an endogenous gene with a transgene while maintaining normal gene expression. In combination with recombineering strategies this technology is useful for RNAi rescue experiments, protein localisation and the identification of protein interaction partners in mammalian tissue culture cells. In summary, this thesis presents a major technical advance for large-scale functional genomic studies in mammalian tissue culture cells and provides novel insights into various aspects of cell cycle progression. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 217 MB: Movies, Rohdaten - Nutzung: Referat Informationsvermittlung der SLUB)

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.

Proteomic analyses of centrosomes from diverse species are helping to identify conserved components of these organelles. Amongst these studies, a group of novel proteins has been identified and postulated as candidate core centriolar components, dubbed Poc proteins, for proteome of centriole. Meanwhile, studies on the Xenopus germ plasm protein, Xpat, led to the identification of an interacting protein, named Pix for protein that interacts with Xpat. Later sequence comparison revealed that Poc1 and Pix are the same protein and will here be referred to as Pix. Pix proteins localize to centrosomes and spindle poles in human cells and the basal bodies of Chlamydomonas and Tetrahymena. It has been suggested that Pix proteins are required for centriole duplication and length control, as well as potentially ciliogenesis. Pix proteins have also been localized to mitochondria in human cells where they might act as molecular adaptors to anchor microtubules to mitochondria. Human cells encode two Pix proteins and the aim of this thesis was to investigate the cell cycle-dependent regulation of Pix1 and Pix2 in human cells. For this purpose, we first generated polyclonal rabbit antibodies that were specific for either Pix1 or Pix2. We then showed for the first time that both Pix isoforms localize to centrosomes throughout the cell cycle and independently from one another. Using the antibodies, we also confirmed that Pix1, but not obviously Pix2, is not an intrinsic mitochondrial protein but localizes to the surface of mitochondria. Through a proteomic approach, we identified two molecular chaperons that potentially interact with Pix proteins, HSP90 and TCP1, and demonstrated that Pix proteins can form dimers. In addition, we produced the first experimental evidence that human Pix proteins are alternatively spliced, suggesting that additional, undiscovered Pix isoforms might be expressed in human cells. Finally, we found that Pix1 is phosphorylated in a mitosis-specific manner, and that this is potentially regulated by Cdk1. Thus, we propose that Pix proteins have a specific and previously unidentified role in human mitotic cell division.

Gene expression is a process that is tightly regulated by many factors. Different genes are transcribed not only in a cell specific manner but are also differentially expressed at different stages of the cell cycle. Cnot3 is part of the CCR4-NOT deadenylation complex, which is involved in the turnover of mRNAs in the cytoplasm and has also been shown to have roles in regulating transcription and cell proliferation and in maintaining ES cell pluripotency. Previous work demonstrated that Cnot3 interacts directly with Aurora B kinase and is phosphorylated by Aurora B in an in vitro assay. Aurora B and Cnot3 co-localise at active gene promoters in resting B cells. Since Aurora B is a cell cycle kinase, I have developed a cell synchronization system to analyse the role of the Cnot3-Aurora B interaction at different stages of the cell cycle in primary B cells. Using this system, I have demonstrated that the interaction between Cnot3 and Aurora B varies during cell cycle progression. In vitro analysis showed that the interaction occurs through the NOT box domain of Cnot3. Mass spectrometry analysis of Cnot3 interactors, performed on nuclear extracts from B cells in the early G1 and G2 phases of the cell cycle, identified interactions with many factors that are known to have roles in transcription regulation and RNA processing. Interaction of Cnot3 with Histone H1 was confirmed using a peptide binding assay, suggesting a potential role in chromatin organization. Cnot3 was also shown to interact with Xrn2, a 5'-3' exoribonuclease that is involved in RNA turnover and termination of transcription. ChIP analysis demonstrated promoter binding of Cnot3 at a number of cell cycle stages. Cnot3 shows cell cycle dependent binding to promoters of a wide range of active genes, including promoters that are not directly involved in cell cycle regulation. Genome wide analysis using ChIP sequencing revealed changes in the binding profiles of Cnot3 at promoters and enhancers during cell cycle progression. A Cnot3 conditional knock out mouse has been generated, which will be used to test the functional importance of these observations.

L'ubiquitylation est une modification post-traductionnelle qui joue de nombreuses fonctions importantes dans les cellules. Au cours de mon doctorat, je me suis concentré sur deux composants du système d'ubiquitine : l'enzyme de déubiquitination (DUB) ubiquitine carboxyl-terminal esterase L3 (UCHL3) et la protéine à domaine de liaison à l'ubiquitine (UBD) ubiquitin associated protein 2 like (UBAP2L, également connue sous le nom de NICE4). Mes études ont montré que l'UCHL3 est nécessaire au maintien de la forme nucléaire des cellules humaines et à la ségrégation des chromosomes. Au niveau moléculaire, UCHL3 interagit physiquement avec Aurora B et la déubiquitylise, régulant ainsi sa localisation au niveau des kinétochores et son interaction avec MCAK. Dans mon deuxième projet, j'ai identifié une nouvelle fonction d'UBAP2L dans la régulation des protéines liées au syndrome de retard mental du X fragile (FXRPs) et dans l'homéostasie des complexes de pores nucléaires (NPCs), qui, étonnamment, est indépendante de la liaison à l'ubiquitine d'UBAP2L. Au lieu de cela, j'ai découvert que les arginines dans le domaine arginine-glycine-glycine (RGG) de UBAP2L sont nécessaires pour l'interaction avec les FXRPs, et médient la fonction sur les FXRPs et les nucléoporines (Nups) au début de la phase G1
Ubiquitylation is a posttranslational modification which plays many important functions in cells. During my PhD study, I focused on two components of the ubiquitin system : the deubiquitinating enzyme (DUB) ubiquitin carboxyl-terminal esterase L3 (UCHL3) and the ubiquitin-binding domain (UBD) protein ubiquitin associated protein 2 like (UBAP2L, also known as NICE4). My studies showed that UCHL3 is necessary for maintaining proper nuclear shape of human cells and chromosome segregation. At the molecular level, UCHL3 physically interacts with and deubiquitylates Aurora B, thereby regulating its localization at the kinetochores and interaction with MCAK. In my second project, I identified a novel function of UBAP2L in the regulation of Fragile X mental retardation syndrome-related proteins (FXRPs) and in the nuclear pore complexes (NPCs) homeostasis, which surprisingly, is independent of UBAP2L ubiquitin-binding. Instead, I found that the arginines within the arginine–glycine–glycine (RGG) domain of UBAP2L are required for the interaction with FXRPs, and mediate the function on FXRPs and nucleoporins (Nups) in early G1 phase

The unicellular eukaryote Trypanosoma brucei causes African Sleeping sickness and multiplies extracellularly in the bloodstream of the infected host. The parasite evades antibody-mediated lysis by switching its Variant Surface Glycoprotein (VSG) coat. Blocking VSG synthesis results in an abrupt growth inhibition and a precise pre-cytokinesis cell cycle arrest, with an accumulation of cells with two nuclei and two kinetoplasts. Additionally, induction of VSG RNAi triggers a global block in translation, which is not due to a general decrease in transcript levels. The mechanism behind this translation arrest was investigated. It was observed that it correlated with a decrease in polysomes, indicating that translation was blocked at the level of initiation. It was also shown that the VSG RNAi-triggered growth inhibition was reversible, which suggests that this is not a lethal phenotype. The VSG221 RNAi-induced growth arrest could be alleviated if a second different VSG (VSG117), which was not recognised by the VSG221 RNAi, was expressed immediately downstream of the promoter of the active VSG221 Expression site. Further, it was possible to delete the telomeric VSG221 in these VSG double-expressors, leaving the cells completely reliant on the second complementing VSG117 gene. VSG117 expressed from a promoter-adjacent position in the active Expression site was shown to form a functional surface coat that protected the parasites from complement-mediated lysis in vitro. Transiently transfecting cells with anti-VSG221 morpholino oligonucleotides allowed us to specifically block translation of VSG221 mRNA without degrading it. This resulted in a pre-cytokinesis cell cycle arrest similar to that induced by VSG221 RNAi. This indicates that the VSG RNAi-triggered growth inhibition was due to a lack of VSG protein or its synthesis rather than the ablation of the abundant VSG mRNA. In addition, it was shown that blocking VSG synthesis reduced the rate of surface VSG internalisation in cells that were stalled precytokinesis, but had no effect on other endocytic markers. These experiments give us further insight into the importance of the protective VSG coat for pathogenicity in T. brucei.

Myosin VI is a unique actin motor involved in multiple biological functions, such as endocytic and secretion processes, cell migration, autophagy, and in the maintenance of the Golgi complex and stereocilia. These functions are dictated by the interaction of myosin VI with different cargos, which can also regulate the ability of this protein to work as an anchor or a motor that moves along actin filaments. Previous experiments performed in our laboratory led to the identification of a novel set of myosin VI interactors that belong to the centrosome compartment, suggesting that myosin VI could have an important and unexpected role in centrosomal processes. Indeed, the depletion of myosin VI leads to alterations in the centrosome structure and number, and to an impairment in the formation of the primary cilium. Our data also suggest a potential role of myosin VI in the regulation of cell cycle progression. Indeed, myosin VI depletion leads to cell cycle arrest and senescence caused by p53 activation. To characterize this phenotype, we performed a genome-wide CRISPR/Cas9 rescue screening, that led us to identify some candidates potentially involved in p53 activation following myosin VI depletion. This study unveils a new role for myosin VI in centrosome biology and in the control of the cell cycle, two processes whose dysregulation is an important step during carcinogenesis.

Embryonic exposure to the anticonvulsant drug Dilantin has been shown to have detrimental effects on development. Some of the observed effects include growth retardation, craniofacial defects, and even death. As the drug is metabolized, toxic intermediates form, which could be causing the characteristic abnormalities observed in embryos exposed to Dilantin. Culture of preimplantation mouse embryos in the presence of IOµg/mL or 20µglmL Dilantin show a slowing of development inl9.3% and 19.1% ofembryos respectively at Day 3 of culture. The toxic intermediates could be causing alterations in cyclin expression, cell cycle proteins, or the cell cycle timing itself. Previous research determined an in vivo baseline expression for cyclins B 1, E, D, A and cdk2, which was used to compare the expression of these cyclins and cdks between in vivo and Dilantin cultured embryos. Altered patterns in cultured embryos suggested that an alteration in cell cycle timing, therefore, S phase timing was determined in cultured untreated embryos utilizing 5'-Bromo-2-deoxyuridine (BrdU) incorporation and indirect immunofluorescence staining. The results of the experiment showed the second S phase was at 30 hpf, approximately 9 hours later, and the third S phase was at 54 hpf, approximately 3 hours later than previous in vivo literature reports. S phase timing in NaOH vehicle controls did not appear different from untreated controls. Dilantin showed S phase peaks at 24 and 55 hpf. In the Dilantin treated embryos, the nuclear staining intensity for the second S phase did not decrease as rapidly as had been observed in control embryos. Embryos that developed beyond the 2-cell stage demonstrated two distinct S phase peaks at 45 and 54 hpf, while embryos at the 2-cell stage did pass through the second S phase but not the third S phase. These data suggested that the Dilantin could be causing a delay in G2. Future experiments would be necessary to determine if the expression of G2 cyclins are being altered in Dilantin treated embryos.
Department of Biology

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.

B lymphopoiesis requires a network of transcription factors that orchestrate changes in gene expression amidst immunoglobulin gene rearrangement and periods of cell proliferation. Although proteins required for the function of this network have been identified, the precise mechanisms that coordinate these processes as hematopoietic progenitors differentiate into lineage-committed B cells remain unclear. Justy mice display a profound arrest of B cell development at the time of lineage commitment due to a point mutation that decreases expression of the protein Gon4-like. Previous studies suggested that Gon4-like functions to coordinate gene expression and cell division to determine cell fate, but the role of Gon4-like in B lymphopoiesis is largely unknown. Here we demonstrate that Gon4-like is required to regulate gene expression and cell cycle progression in B cell progenitors. Expression of genes required for B cell development is intact in Justy B cell progenitors, yet these cells fail to repress genes that promote the development of alternative lineages. In addition, Justy B cell progenitors are unable to upregulate genes that instruct cell cycle progression. Consistent with this, B cell progenitors from Justy mice show signs of impaired proliferation and undergo apoptosis despite containing elevated levels of activated STAT5, a transcription factor that promotes cell proliferation and survival. Genetic ablation of p53 or retroviral-mediated overexpression of pro-survival factors failed to rescue these defects. In contrast, overexpression of proteins that promote the G1/S transition of the cell cycle, including D-type cyclins, E2F2 and cyclin E, rescued pro-B cell development from Justy progenitors, an effect that was not observed upon overexpression of proteins that function during the S and G2M phases of the cell cycle. Further, overexpression of cyclin D3 led to partial restoration of gene repression in Justy pro-B cells. Notably, Gon4-like interacted with STAT5 when overexpressed in transformed cells, suggesting Gon4-like and STAT5 function together to activate expression of STAT5 target genes. Collectively, our data indicate that Gon4-like is required to coordinate gene repression and cell cycle progression during B lymphopoiesis.

The primary cilium, once considered an evolutionary vestige, has re-emerged as an essential organelle for the normal functioning of a wide variety of cellular processes. It is now clear that it has important roles in the control of cell proliferation and signalling during development, with defects in primary cilia being a major cause of many human diseases. These disorders, collectively referred to as ciliopathies, include the cystic kidney diseases that are characterized by aberrant cell proliferation and cyst formation. Mutations in the NIMA-related kinase, Nek8, are associated with cystic kidney disease in both humans and mice, with Nek8 being the candidate NPHP9 gene in the human juvenile cystic kidney disease, nephronophthisis. Previous localisation studies have shown that Nek8 localises to centrosomes and cilia in dividing and ciliated cells, respectively, strengthening the ciliary hypothesis of cystic kidney disease. However, the role of Nek8 in ciliogenesis remains to be defined and no substrates for this kinase have yet been identified. In this thesis, I present data from a series of biochemical and cell biology experiments aimed at investigating the regulation and function of Nek8 with respect to cell cycle progression and ciliogenesis. I first of all show that localization of Nek8 to centrosomes and cilia is dependent upon both its kinase activity and its C-terminal non-catalytic RCC1 domain. Interestingly, Nek8 was capable of phosphorylating the RCC1 domain, which in isolation also localized to centrosomes and cilia. This leads us to propose that centrosome recruitment is mediated by the RCC1 domain, but requires a conformational change in the full-length protein that is promoted by autophosphorylation. I then established conditions required for measurement of Nek8 catalytic activity. Besides confirming the predicted activity of catalytic site mutants, this revealed that the three NPHP9-associated point mutants did not exhibit loss of activity. However, as one of these mutants, H425Y, failed to localize correctly, we predict that this mutation, which lies in the RCC1 domain, alters the RCC1 conformation such that it disrupts its centrosome targeting motif. Importantly, I also show that serum starvation induces proteasomal degradation of Nek8, specifically in cell lines in which serum starvation induces quiescence and ciliogenesis. Strikingly, serum starvation also induces Nek8 kinase activity, whilst maintained expression of Nek8 appears to suppress ciliogenesis. Taken together, these findings not only reveal important mechanisms through which Nek8 activity and localization are regulated, but suggest that Nek8 itself may have both positive and negative activities in the process of ciliogenesis.

MED12 is an X– chromosome member of the Mediator complex that is a key regulator of tissue specific gene expression and moderates intracellular signaling via multiple developmental pathways. Sequence variation in the carboxy– terminus of MED12, which contains a PQL and Opa domain, is associated with X– linked mental retardation behavioral syndromes and schizophrenia. Unfortunately, the mechanism(s) through which sequence variation in the carboxy– terminus could alter vulnerability to neurodevelopmental and neuropsychiatric illnesses is yet unclear. In order to elucidate a better understanding of this process, we examined the role of the MED12 carboxy– terminus in cell cycle and gene expression with a full– length overexpression construct, domain deleted overexpression constructs and RNA interference using a HEK293 cell model. Our results show that MED12 overexpression leads to G1 cell cycle exit, whereas deletion of the PQL domain and MED12 RNA interference results in cell cycle progression. Our data also show that MED12 expression level differentially affects early response antiviral gene expression and stress response mechanisms. These results are consistent with prior studies showing that MED12 has a key role in determining neuronal cell fate and with the theoretical understanding of the biological basis of psychosis. These results also lend further insight upon the pathways through which MED12 exerts its effects upon differentiation and disease pathogenesis, which may lead to new approaches to the treatment of MED12– related disorders.

SRY (sex determining region Y)-box 2 (SOX2) is one the embryonic stem cell transcription factors that is capable of reprogramming adult differentiated cells into an induced pluripotent cell. SOX2 is amplified in various types of epithelial cancers and its high its expression correlates with poor prognosis and decreased patient survival. Aberrant Wnt signaling drives the colo-rectal carcinogenic process and is a major determinant of the disease outcome. This study demonstrates that SOX2 counteracts Wnt driven tumor cell proliferation and maintains quiescence in a sub-population of Colo-Rectal Cancer (CRC) cells. High SOX2 expression is found in a sub-group of CRC patients with advanced disease. High SOX2 expression coupled with low Wnt activity was found in SW620 metastatic CRC cell line, while the opposite was true for the isogenic SW480 primary tumor cell line. SOX2 silencing increased Wnt activity and enhanced the oncogenic potential of SW620 cells in vitro and in vivo while over-expression had opposite effects in SW480 cells. SOX2 up-regulates the expression of PTPRK and PHLPP2 protein phosphatase genes which in turn attenuates Wnt activity by interfering with Protein Kinase A, B and C mediated beta catenin phosphorylation at Serine 552 and 675 amino acid residues thereby diminishing its nuclear sequestration and transcriptional activation. Thus SOX2 mitigates growth factor mediated Wnt activation in CRC cells and inhibits cellular proliferation so that these cells are forced to change their oncogene addiction. In effect, high SOX2 expression causes clonal evolution of APC mutant CRC cells from a state of high Wnt dependency to a state of low Wnt dependency in the process making such cells resistant to Wnt inhibitor therapy. Enhanced SOX2 transcriptional activity was associated with increased proportion of cancer cells in G0-G1 phase of cell cycle. Changing SOX2 protein levels in cells had a direct correlation with mRNA levels of RBL2-HUMAN and CDKN2B genes, which serve as regulators of G0 and G1 respectively. SOX2 was shown to physically bind and to the promoter region of these two genes and enhance their transcription. Thus high SOX2 expression, up-regulates the expression of key cell cycle inhibitor genes like RBL2 and CDKN2B and keeps cells in a dormant state. This phenomenon allows colon cancer cells to escape from cytotoxic drug therapy directed at rapidly dividing cells and cause treatment failure and disease relapse.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
The complex [alpha]-proteobacterial cell cycle regulatory network is essential not only for faithful replication and segregation of the genome, but also to coordinate unique cellular differentiation events that have evolved as adaptations to the different lifestyles of this diverse group of bacteria. The soil-dwelling [alpha]-proteobacterium, Sinorhizobium meliloti, not only has to accurately coordinate the replication of its tripartite genome, but also must undergo a dramatic cellular differentiation in order to form an effective symbiosis with the legume Medicago sativa. Preliminary analyses have indicated that plasticity in the S. meliloti cell cycle regulatory network may be essential to symbiosis, but cell cycle research in S. meliloti has been hindered largely by lack of a method to obtain synchronous populations of S. meliloti. In this thesis, I present the first method to generate synchronous cultures of S. meliloti. I performed microarray gene expression analysis on synchronous populations of S. meliloti to gain a global view of transcriptional regulation of cell cycle events. This represents the first work of this kind done in an [alpha]-proteobacterium besides Caulobacter crescentus, which is the current model for [alpha]-proteobacterial cell cycle studies. The importance of transcriptional regulation of cell cycle progression was first discovered in C. crescentus and the work presented in this thesis highlights the conservation of cell cycle regulated gene expression in S. meliloti. I identified 462 cell cycle regulated transcripts in S. meliloti, which included genes involved in vital cell processes such as cell division, flagella biogenesis, replication and segregation of its tripartite genome as well as several putative cell cycle regulators. I compared the set of genes with cell cycle regulated transcripts identified in my analysis with the set identified in C. crescentus to generate a core set of 128 conserved genes demonstrating cell cycle regulated gene expression in both species. To determine which of the S. meliloti genes with cell cycle regulated transcripts might be part of the CtrA and DnaA regulons in S. meiloti, I performed CtrA and DnaA binding motif analysis. To understand the evolutionary significance of these CtrA and DnaA binding motifs, I looked at conservation of these motifs in homologous genes from several related [alpha]-proteobacteria. The results indicated that the putative CtrA regulon might be more evolutionarily constrained than the putative DnaA regulon. Organisms more closely related to S. meliloti or with more similar lifestyles demonstrated a much greater conservation of the CtrA binding motifs identified in S. meliloti. The CtrA binding motifs in S. meliloti identified by my analysis were not at all well conserved in C. crescentus, which was the most distantly related [alpha]-proteobacteria surveyed. These differences in cell cycle regulated transcription and the putative CtrA regulon between S. meliloti and C. crescentus thus appear to represent specific adaptations to the distinctive genome and unique intracellular symbiotic lifestyle of S. meliloti and illustrate the importance of S. meliloti as a model for cell cycle regulation in [alpha]-proteobacteria with similar intracellular lifestyles. The work presented in this thesis also describes the importance of CtrA regulation in S. meliloti during symbiosis with M. sativa. A crucial part of this symbiosis is a striking cellular differentiation (termed bacteroid differentiation), which includes changes in membrane permeability, cell elongation and branching, endoreduplication of the genome and loss of reproductive capacity and therefore a significant deviation from the free-living cell cycle program. Endoreduplication of the genome requires a decoupling of DNA replication and cell division, which could be achieved by down-regulation of the essential master cell cycle regulator CtrA. I tested the effects of CtrA depletion in S. meliloti and found that CtrA depletion induces a bacteroid-like state characterized by elongated and branched cells and highly elevated DNA content. I also show that S. meliloti CtrA has a comparable half-life to C. crescentus CtrA, but regulated proteolysis of CtrA may be different in the two species since we found CtrA proteolysis to be essential in S. meliloti. In addition, I demonstrate that the promoter and coding regions of C. crescentus ctrA cannot complement an S. meliloti ctrA chromosomal deletion during symbiosis even though they can do so in the free-living state. My attempts to identify the defects in the function C. cresentus ctrA promoter or coding region within M. sativa gave surprising results since S. melioti strains expressing C. crescentus CtrA from the S. meliloti ctrA promoter region and vice versa were able to establish an effective symbiosis with M. sativa. I discuss several possibilities to explain this apparent paradox, but further study is required to fully clarify this observation. Taken as a whole, my thesis work represents a significant advancement to the field of cell cycle research in S. meliloti and [alpha]-proteobacteria as a whole. The cell synchronization method I developed will greatly facilitate more comprehensive analysis of cell cycle regulation in S. meliloti. My microarray gene expression analysis provides a global view of cell cycle regulated transcription in S. meliloti, which can be used in more in-depth explorations of specific mechanisms of transcriptional regulation of cell cycle events in S. meliloti. Lastly, my study of CtrA function in S. meliloti establishes the importance of CtrA regulation during symbiosis with M. sativa.
by Nicole J. De Nisco.
Ph.D.