In order to generate healthy daughter cells, nuclear division and cytokinesis need to be coordinated. Premature division of the cytoplasm in the absence of chromosome segregation or nuclear proliferation without cytokinesis might lead to aneuploidy and cancer. The cyclin dependent kinases, CDKs, are a main regulator of the cell cycle. Timely increase and decrease in their activity is required for cell cycle progression. To enter mitosis, mitotic CDK activity needs to rise. CDK activity stays elevated until chromosome segregation is completed and exit from mitosis requires decrease in CDK activity. Observations in several experimental systems suggest that coordination of cytokinesis with the nuclear cycle is regulated through CDK activity. Prolonged high CDK activity, as it occurs when chromosome segregation is delayed, was found to oppose cytokinesis. Prevention of cytokinesis through high CDK activity may therefore provide a mechanism to prevent precocious cell division in the absence of chromosome segregation. To prevent polyploidy when cell division is delayed, progression through the next nuclear cycle should be inhibited until cytokinesis is completed, presumably by the inhibition of CDK activity. In the fission yeast Schizosaccharomyces pombe, a signaling cascade called Septation Initiation Network (SIN) is required for the coordination of cytokinesis with the nuclear cycle. The SIN is essential for cytokinesis, triggering the execution of cell division through constriction of the actomyosin ring. The activation of the SIN signaling cascade, and thus cytokinesis, is opposed by high CDK activity, preventing precocious cytokinesis. S. pombe delay entry into the next nuclear division in response to delayed cytokinesis due to defects in the contractile ring until cytokinesis is completed thereby preventing the accumulation of multinucleate, non viable cells. This safeguard against multinucleate cells is termed the cytokinesis checkpoint. The cytokinesis checkpoint keeps CDK activity low, preventing nuclear cycle progression. The SIN is required for the cytokinesis checkpoint and therefore is a key coordinator between nuclear cycle and cytokinesis. How the SIN functions in the cytokinesis checkpoint was not known. Cdc14-family phosphatases are highly conserved from yeast to humans, but were only characterized in Saccharomyces cerevisiae at the time this thesis was initiated. Cdc14 had been identified as the effector of a signaling cascade homologous to the SIN, called the mitotic exit network (MEN), which is required for exit from mitosis. This thesis describes the identification of the S. pombe Cdc14-like phosphatase Clp1p as a component of the cytokinesis checkpoint. Clp1p opposes CDK activity, and Clp1p and the SIN activate each other in a positive feedback loop. This maintains an active cytokinesis checkpoint and delays mitotic entry. We further found that Clp1p regulates chromosome segregation. Concluding, this thesis describes discoveries adding to the characterization of the cytokinesis checkpoint and the function of Clp1p. While others found that Cdc14-family phosphatases, including Clp1p, have similar catalytic functions, we show that their biological function may be quite different between organisms, possibly due to different biological challenges.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-115).

Thesis (Ph. D.)--University of South Florida, 2004. / Includes vita. Includes bibliographical references (leaves 124-148).

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 173-176.

The DNA replication checkpoint transcriptionally up-regulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF (aka DSC1), the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitrophosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Substitution of two potential phosphorylation sites with phospho-mimetic amino acids suffice to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggest that checkpoint regulation of the MBF transcription factor may be a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G1/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes. Our result shows that both the replication checkpoint and the S-phase DNA damage checkpoint are involved in activating MBF regulated S-phase gene transcription and that this coordinated transcriptional response is beneficial for survival during replication stress. I demonstrate that the beneficial role of the transcriptional response during checkpoint activation is mediated by three major MBF transcripts: cdc22, mrc1 and mik1. Mrc1 dependent stabilization of stalled fork is important during S phase arrest. However, cells ability to prevent mitosis (Mik1 dependent) along with stable fork (Mrc1 dependent) both are crucial for survival. Our data also suggest that the level of Cdc22 is a determining factor for replication checkpoint activation and when over-expressed can alleviate the effects not only in HU but also in MMS.

Mobile genetic elements represent a large portion of the genome in many species. Posing a danger to the integrity of genetic information, silencing and structural machinery has evolved to suppress the mobility of foreign and transposable elements within the genome. Condensin proteins – which regulate chromosome structure to promote chromosome segregation – have been demonstrated to function in repetitive gene regulation and transposon silencing in several species. In model system Caenorhabditis elegans, microarray analysis studies have implicated Condensin II subunit HCP-6 in the silencing of multiple loci, including DNA transposon MIRAGE. To address the hypothesis that HCP-6 has a direct function in transcriptional gene silencing of the MIRAGE transposon, we queried MIRAGE expression and chromatin profiles in wild-type and hcp-6 mutant animals. Our evidence confirms that HCP-6 does indeed function during silencing of MIRAGE. However, we found no significant indication that HCP-6 binds to MIRAGE, nor that HCP-6 mediates MIRAGE enrichment of H3K9me3, the repressive heterochromatin mark observed at regions undergoing transcriptional silencing. We suggest that the silencing of MIRAGE, a newly evolved transposon and the only tested mobile element considerably derepressed upon loss of HCP-6, is managed by HCP-6 indirectly.

Histone Nuclear Factor P (HiNF-P) is a known transcriptional regulator that is critical for the activation of replication dependent histone H4 genes during S phase. HiNF-P is a 65 kDa zinc finger protein that binds to its consensus binding sequence in the Cell Cycle Control Element (Site II) of the proximal promoter region of 11 of the 14 histone H4 genes. HiNF-P is a known co-factor of the global histone gene regulator and cyclinE/CDK2 substrate p220NPAT, however it was not known if this regulatory function reflected a physical interaction. In addition, other HiNF-P interacting proteins have yet to be identified. The work presented in this thesis identifies and characterizes HiNF-P interactions with various proteins within the cell, including p220NPAT. A yeast two-hybrid interaction screen identified candidate interacting proteins of HiNF-P and provided insight into novel cellular functions and transcriptional targets. A candidate yeast two-hybrid approach identified an interaction between HiNF-P and p220NPAT. This direct physical interaction links the cyclin E/CDK2 signaling pathway governing the G1/S phase transition with replication dependent histone gene transcription in S phase. An unbiased yeast two-hybrid screen for HiNF-P interacting proteins revealed an interactome library which suggests roles of HiNF-P in multiple cellular processes. This screen identified 67 candidate HiNF-P interacting proteins that are RNA processing factors, known and putative gene regulators, uncharacterized proteins, proliferation related proteins, as well as metabolic and signaling proteins. Identification of multiple RNA binding and processing factors, including the splicing cofactor, SRm300, links HiNF-P to mRNA processing. HiNF-P is potentially functioning in mRNA processing by interacting with these proteins directly and functioning in complex with them, or more likely, by recruiting these and other splicing factors to sites of transcription. We identified a number of known and putative gene regulators which are candidate HiNF-P interacting proteins. We isolated the atypical C2CH zinc finger protein, THAP7, a known transcriptional repressor. THAP7 interacts with HiNF-P by co-immunoprecipitation and co-immunofluorescence experiments. We show forced expression of THAP7 abrogates HiNF-P/p220 mediated activation of histone H4 gene transcription. THAP7 may represent a novel co-factor of HiNF-P and p220 mediated regulation of histone H4 genes. Identification of interacting proteins of HiNF-P that are involved in transcriptional regulation provides insight into other transcriptional targets of HiNF-P. HiNF-P is localized throughout the nucleus, presumably at multiple gene foci. These interacting proteins may represent novel co-factors of HiNF-P regulation of these other multiple target genes. HiNF-P has been identified as a regulator of cell cycle dependent histone genes, therefore we were interested in identifying other proliferation related proteins with which HiNF-P is interacting. We identified a number of proteins thought to be involved in cellular proliferation, including Ki-67 and an unknown protein XTP2. The functions of these proteins have not been identified. An interaction with HiNF-P might suggest a role for these proteins in histone gene regulation. In addition, Ki-67 has been implicated transcriptional control of ribosomal genes, although no role of HiNF-P in this function has been identified. HiNF-P is a known regulator of histone gene expression via a functional interaction with the global histone gene regulator and cyclin E/CDK2 substrate, p220. This thesis demonstrates HiNF-P directly interacts with the N-terminus of p220. This interaction requires multiple regions within the N-terminus including a LisH-like domain known to function in protein-protein interactions, a region (aa 121-145) known to be required for histone gene transactivation, and another uncharacterized region (209-318). In addition a phylogenically conserved region within the C-terminus of HiNF-P, the HiNF-P Specific Conserved Region (PSCR) is necessary for this interaction. Mutational analysis of these regions abrogates this interaction. HiNF-P and p220 co-localize at specific foci within the cell corresponding to Cajal bodies, which are known sites of histone gene clusters. This work shows that this interaction is necessary for histone gene transcriptional activation and HiNF-P dependent recruitment of p220 to histone H4 gene promoters. In addition HiNF-P as well as p220 interact with the Stem Loop Binding Protein (SLBP) and co-localize in situ. SLBP is a necessary factor for histone pre-mRNA processing events which also occur at Cajal bodies. These interactions provide evidence of the coupling of transcription and processing of histone genes and the involvement of common factors in both processes. This would allow for rapid production of abundant histone proteins which is needed during S phase. This thesis has identified multiple candidate interacting proteins of HiNF-P. These proteins establish HiNF-P as a protein involved in many cellular processes and mechanisms beyond transcriptional control of cell cycle dependent histone genes.

Cytokinesis is the final event of the cell division cycle and results in physical and irreversible separation of a mother cell into two daughter cells. Cytokinesis must only occur after chromosomes have segregated during mitosis to ensure each daughter cell receives the proper complement of genetic material. Failure to execute normal cytokinesis can result in aneuploidy and/or polyploidy, a hallmark of many cancers. Cytokinesis occurs mechanically through constriction of an actin-myosin based contractile ring, while initiation of ring constriction is temporally and spatially mediated by complex signaling networks. It is absolutely crucial that cytokinesis is tightly coordinated with the cell cycle in order to preserve the fidelity of cell division. We hypothesized that to achieve such tight control of cytokinesis, cells may utilize both promotional and inhibitory signals, however how cells maintained this control was poorly understood. The goal of this thesis was to characterize how cells regulate signaling of cytokinesis, both positively and negatively, during cell division using the fission yeast Schizosaccharomyces pombe as a model organism. Two approaches were employed. (1) We first sought to characterize the positive timing mechanism that signals cytokinesis though a detailed investigation of Sid1p, a protein kinase essential for activation of ring constriction. (2) Secondly, we sought to define how cells negatively regulate cytokinesis through investigation of Dma1p, a spindle checkpoint protein implicated in inhibition of cytokinesis. Our results reveal a conserved signaling network, termed the Septation Initiation Network (SIN), of which Sid1p is an intermediate component, that controls temporal and spatial regulation of cytokinesis. We found Sid1p is additionally controlled by Cyclin Dependent Kinase activity, uncovering an important link between mitotic events and initiation of cytokinesis. Furthermore, we found that aberrant SIN activation can override a microtubule-damage-induced spindle checkpoint arrest. This effect is counteracted by Dma1p, which normally inhibits the SIN during checkpoint activation to preserve cell viability until damage is repaired. We conclude that signaling cytokinesis is tightly coordinated with mitosis in S. pombe by positive signals acting through Sid1p and the SIN, and under certain conditions, negative signals acting through Dma1p. Considering the conservation of cell cycle regulators in the eukaryotic kingdom, it is likely that similar mechanisms to control cytokinesis exist in humans.

The fission yeast Schizosaccharomyces pombe has become a powerful model system for studying cytokinesis, a process of cytoplasmic division by which one cell divides into two identical daughter cells. Like mammalian cells, S. pombe divides through the use of an actomyosin contractile ring, which is composed of a set of highly conserved cytoskeletal proteins. Cytokinesis in S. pombe is primarily regulated by the SIN pathway, which is activated in late mitosis and is required for actomyosin contractile ring and septum assembly, and also plays a role in spindle checkpoint inactivation, and telophase nuclear positioning. The various functions of the SIN are carried out by the terminal kinase in the pathway called Sid2. The lack of information in the downstream targets of Sid2 has limited our understanding of the different functions of the SIN. We recently showed that, in addition to its other functions, the SIN promotes cytokinesis through inhibition the MOR signaling pathway, which normally drives cell separation and initiation of polarized growth following completion of cytokinesis (Ray et al, 2010). The molecular details of this inhibition and the physiological significance of inhibiting MOR during cytokinesis was unclear. The results presented in Chapter II describe our approach to identify Sid2 substrates, particularly focusing on Nak1 and Sog2 that function in the MOR signaling cascade. We identified and characterized Sid2 phosphorylation sites on the Nak1 and Sog2 proteins. Chapter III explores how post translational modification of MOR proteins by Sid2 regulates polarized growth during cytokinesis. This includes delineating the effect of Sid2 mediated phosphorylation of Nak1 and Sog2 on protein-protein interactions in the MOR pathway as well as on the regulation of their localization during late mitosis. Finally, results in Chapter IV demonstrate that failure to inhibit MOR signaling is lethal because cells initiate septum degradation/cell separation before completing cytokinesis thereby emphasizing the importance of cross-regulation between the two pathways to prevent initiation of the interphase polarity program during cytokinesis.