Investigating the arrangement of the packed DNA inside the nucleus has revealed the essential role of genome organisation in controlling genome function. Furthermore, genome architecture is highly dynamic and significant chromatin re-organisation occurs in response to environmental changes. However, the mechanisms that drive the 3D organisation of the genome remain largely unknown. To understand the effect of biophysical properties of chromatin on the dynamics and structure of chromosomes, I developed a 3D computational model of the nucleus of the yeast S. cerevisiae during interphase. In the model, each chromosome was a hetero-polymer informed by our bioinformatics analysis for heterogeneous occupancy of chromatin-associated proteins across the genome. Two different conditions were modelled, normal growth (25°C) and heat shock (37°C), where a concerted redistribution of proteins was observed upon transition from one temperature to the other. Movement of chromatin segments was based on Langevin dynamics and each segment had a mobility according to their protein occupancy and the expression level of their corresponding genes. The model provides a significantly improved match with quantitative microscopy measurements of telomere positions, the distributions of 3D distances between pairs of different loci, and the mean squared displacement of a labelled locus. The quantified contacts between chromosomal segments were similar to the observed Hi-C data. At both 25°C and 37°C conditions, the segments that were highly occupied by proteins had high number of interactions with each other, and the highly transcribed genes had lower contacts with other segments. In addition, similar to the experimental observations, heat-shock genes were found to be located closer to the nuclear periphery upon activation in the simulations. It was also shown that the determined distribution of proteins along the genome is crucial to achieve the correct genome organisation. Hence, the heterogeneous binding of proteins, which results in differential mobility of chromatin segments, leads to 3D self-organisation.

Being able to respond to extracellular cues such as nutrients and growth factors is of vital importance to all living cells. Pathways have therefore evolved which can sense the extracellular status, transmit a signal through the cell and affect gene expression, which ultimately enables adaptation. Intriguingly, research has revealed that such signaling pathways responding to nutrient status are intrinsically linked to the lifespan of organisms, a phenomenon known as caloric restriction. This thesis utilizes budding yeast, Saccharomyces cerevisiae, as a model system to investigate how transcription factors affect gene expression in response to nutrient signaling pathways. Paper I investigates the role of the three homologous transcription factors Mig1, Mig2 and Mig3 in regulating gene expression in response to glucose. This is done by transcriptional profiling with microarrays of wild type yeast, as well as mutant strains where the MIG1, MIG2 and MIG3 genes have been deleted in all possible combinations. The results reveal that Mig1 and Mig2 act together, with Mig1 having a larger effect in general while Mig2 has a role specialized for high-glucose conditions. Using a strategy similar to that in paper I, paper II examines the roles of the two homologous transcription factors Gis1 and Rph1 in gene regulation. This study shows that Gis1 and Rph1 are both involved in nutrient signaling, acting in parallel with a large degree of redundancy. Furthermore, we find that these two transcription factors change both target genes as well as the effects on transcription when the yeast cell transitions through different growth phases. Rph1 is a functional JmjC histone demethylase, and paper III investigates the connection between this activity and the transcriptional regulation studied in paper II. We find that rendering Rph1 catalytically inactive has little effect on its role in nutrient signaling and gene regulation, but subtly affects certain groups of genes. Paper IV reveals that Rph1 does not affect the chronological lifespan of yeast as does its homolog Gis1. However, deleting or overexpressing RPH1 has effects on the response to rapamycin and caffeine, inhibitors of the evolutionary conserved TORC1 complex affecting lifespan in both yeast and mammals.

Aberrant DNA synthesis and maintenance have been implicated in numerous human diseases. I describe here a novel strategy for systematically identifying budding yeast mutants with elevated levels of DNA damage foci, which represent hubs of DNA damage and repair. A previous study manually scored foci in single mutants but was limited in its ability to survey many conditions in large populations. I developed an automated and statistically robust method for identifying aberrant foci phenotypes by combining synthetic genetic array (SGA) and high-content screening (HCS) methodology. Using this approach, I scored thousands of essential and non-essential gene mutants subjected to environmental and genetic perturbations, including the DNA damaging agent, phleomycin, and deletions of DNA repair genes, SGS1 and YKU80. Collectively, I identified a functionally enriched set of 367 mutants that had increased frequencies of DNA damage foci and established SGA-HCS as a powerful tool for investigating the yeast DNA damage response.

Aberrant DNA synthesis and maintenance have been implicated in numerous human diseases. I describe here a novel strategy for systematically identifying budding yeast mutants with elevated levels of DNA damage foci, which represent hubs of DNA damage and repair. A previous study manually scored foci in single mutants but was limited in its ability to survey many conditions in large populations. I developed an automated and statistically robust method for identifying aberrant foci phenotypes by combining synthetic genetic array (SGA) and high-content screening (HCS) methodology. Using this approach, I scored thousands of essential and non-essential gene mutants subjected to environmental and genetic perturbations, including the DNA damaging agent, phleomycin, and deletions of DNA repair genes, SGS1 and YKU80. Collectively, I identified a functionally enriched set of 367 mutants that had increased frequencies of DNA damage foci and established SGA-HCS as a powerful tool for investigating the yeast DNA damage response.

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

No description available.

<p>Intracellular membrane trafficking is regulated by a large number of protein complexes and lipids. Blocking of trafficking disrupts normal membrane dynamics and causes membrane stress. Two similar proteins from <i>Saccharomyces cerevisiae</i>, Myr1 and Smy2, each containing a polyproline-binding GYF domain, were discovered in separate screens for dosage suppressors of trafficking mutations. The functions of GYF domain proteins are poorly described despite its determined structure and a number of known polyproline peptide ligands. We predicted, using computational analysis, associations between mRNA decay factors and both Myr1 and Smy2, and further demonstrated that they localize to sites of mRNA degradation upon stress, in a GYF domain dependent manner.</p><p>Ypt6 is a small GTPase that regulates vesicle docking at the late Golgi in budding yeast. Myr1 was found as a novel suppressor during the screening of a genomic library in a null ypt6 mutant. Myr1 additionally was capable of rescuing the temperature sensitive growth of a Ric1 deficient strain. Importantly, Ric1 is an activator of Ypt6 and is synthetic lethal with Myr1. Biochemical characterization of the Myr1 protein revealed a limited solubility and an ability to bind cellular membranes, likely relevant to the rescue of trafficking mutants.</p><p>We further assayed the affinity of Myr1 domains to liposomes of distinct composition. Preference for negatively charged lipids suggested possible electrostatic interactions with polybasic clusters within C-terminal regions of Myr1. In contrast, the N-terminus with the GYF domain was found to be capable of self-association. Membrane stress caused by a lipid-bilayer perturbing drug resulted in induced formation of mRNA processing bodies. Cumulatively, these studies suggest that Myr1 functions in the regulation of mRNA stability via its GYF domain, and can sense membrane stress by binding to the lipid bilayer.</p>

Intracellular membrane trafficking is regulated by a large number of protein complexes and lipids. Blocking of trafficking disrupts normal membrane dynamics and causes membrane stress. Two similar proteins from Saccharomyces cerevisiae, Myr1 and Smy2, each containing a polyproline-binding GYF domain, were discovered in separate screens for dosage suppressors of trafficking mutations. The functions of GYF domain proteins are poorly described despite its determined structure and a number of known polyproline peptide ligands. We predicted, using computational analysis, associations between mRNA decay factors and both Myr1 and Smy2, and further demonstrated that they localize to sites of mRNA degradation upon stress, in a GYF domain dependent manner. Ypt6 is a small GTPase that regulates vesicle docking at the late Golgi in budding yeast. Myr1 was found as a novel suppressor during the screening of a genomic library in a null ypt6 mutant. Myr1 additionally was capable of rescuing the temperature sensitive growth of a Ric1 deficient strain. Importantly, Ric1 is an activator of Ypt6 and is synthetic lethal with Myr1. Biochemical characterization of the Myr1 protein revealed a limited solubility and an ability to bind cellular membranes, likely relevant to the rescue of trafficking mutants. We further assayed the affinity of Myr1 domains to liposomes of distinct composition. Preference for negatively charged lipids suggested possible electrostatic interactions with polybasic clusters within C-terminal regions of Myr1. In contrast, the N-terminus with the GYF domain was found to be capable of self-association. Membrane stress caused by a lipid-bilayer perturbing drug resulted in induced formation of mRNA processing bodies. Cumulatively, these studies suggest that Myr1 functions in the regulation of mRNA stability via its GYF domain, and can sense membrane stress by binding to the lipid bilayer.

The Sli15-Ipl1-Bir1 chromosomal passenger complex is essential for proper kinetochore-microtubule attachment and spindle stability in the budding yeast Saccharomyces cerevisiae. Subcellular localization of this complex during anaphase is regulated by the Cdc14 protein phosphatase, which is kept inactive in the nucleolus until anaphase onset. I show here that the predominantly nucleolar ribosome biogenesis protein Utp7 is also present at kinetochores and is required for normal organization of kinetochore proteins and proper chromosome segregation. Utp7 associates with and regulates the localization of Sli15 and Cdc14. It prevents the abnormal localization of Sli15 on cytoplasmic microtubules, the premature concentration of Sli15 on the pre-anaphase spindle, and the premature nucleolar release of Cdc14 before anaphase onset. Utp7 regulates Sli15 localization not entirely through its effect on Cdc14. Furthermore, the mitotic exit block caused by Cdc14 inactivation is relieved partially by the simultaneous inactivation of Utp7. Thus, Utp7 is a multifunctional protein that plays essential roles in the vital cellular processes of ribosome biogenesis, chromosome segregation and cell cycle control. Protein phosphatase 1, Glc7 opposes in vivo functions of the Ipl1-Sli15-Bir1 kinase complex in budding yeast. I show here Scd5- a targeting subunit of Glc7 that regulates endocytosis/cortical actin organization and undergoes nuclear-cytoplasmic shuttling- is present at kinetochores. Ipl1 associates with both Glc7 and Scd5. The scd5-PP1[Delta]2 mutation, which disrupts the association between Glc7 and Scd5, also disrupts the association between Ipl1 and Scd5-Glc7 without affecting the kinetochore localization of these proteins. Genetic studies suggest that Scd5 may positively regulate both Glc7 phosphatase and the Ipl1 kinase complex. In accordance, Scd5 stimulates in vitro kinase activity of Ipl1. scd5-PP1[Delta]2 cells missegregate chromosomes severely due to several defects: i) at least one of sister kinetochores appears not attached to microtubule. ii) sister chromatids are persistently cohesed through anaphase. iii) Sli15 is hyperphosphorylated and less abundant on the anaphase spindle resulting in unstable mitotic spindle. These results together suggest that Scd5 functions in diverse processes that are essential for faithful chromosome segregation. How Scd5 coordinately regulates two apparently antagonistic enzymatic activities of Ipl1 and Glc7 remains to be determined. / text

Among the various external signals perceived by yeast cells, nutrient availability is a condition to which these cells show a highly diverse biological response. Diploid cells in response to different nutritional stress conditions shows different developmental outcomes. On nitrogen starvation, cells undergo dimorphic transition whereby a unicellular yeast form transforms to a multicellular pseudohyphal form. While in the complete absence of a nitrogen source and a fermentable carbon source, yeast cells enter into a complex developmental program termed sporulation which culminates in haploid spores. The main objective of this work was to understand the role played by S. cerevisiaeSUB1 in starvation-induced meiotic program of diploid cells, decipher its target in sporulation specific gene expression cascade, study the domain architecture of Sub1 and examine its functional homology to mammalian PC4. Role of Sub1 in induction of sporulation and other stress responses in S. cerevisiae In a previous whole-genome screen for mutants with altered sporulation efficiency in the Saccharomyces cerevisiae S288c strain, SUB1 locus was identified as a negative regulator of sporulation (Deutschbaueret al., 2002). Moreover, genome-wide gene expression analysis in SK1 strain had shown that SUB1 transcript levels are repressed during sporulation (Chu et al., 1998). Many studies in different yeast strain backgrounds implicate more than 1,000 genesout of 6,200 genes in yeast genome as being differentially expressed during the sporulation process (Chu et al., 1998; Primiget al., 2000; Deutschbaueret al., 2002). Interestingly, these studies show the number of regulatory genes that negatively affect sporulation is far lower than those that are activators of sporulation and moreover their mechanism of action is poorly studied. S. cerevisiae.SUB1 is one among negative regulators of sporulation(Deutschbaueret al., 2002). Global transcriptome of diploid yeast cells undergoing sporulation showed SUB1 transcripts are greatly reduced with time progression (Chu et al., 1998). To understand the role of SUB1 in sporulation, we generated deletion of both SUB1 alleles in the diploid S288c strain background and compared the kinetics of asci formation in this strain with that of the wild-type. We observed that cells lacking SUB1 exhibit ~5-fold increase in tetrad asci. Based on Eosin Y and Calcoflour White staining assays, we find no change in spore morphology in the mutant. Thus the increase in sporulation efficiency in sub1/sub1diploids is not accompanied by formation of defective spores. We validated the reduction in SUB1 transcript levels during sporulation in wild-type SK1 strain background. We also examined the Sub1 protein levels by epitope-tagging of the chromosomal SUB1 open reading frame and determining protein levels in this strain. We find that consistent with the data on transcript levels, Sub1-TAP tagged protein levels too decreased gradually on shift to sporulation medium. We created sub1alleles in diploids in the SK1 strain background and using this strain background we investigated Sub1 target genes and chose IME2 (early), SMK1, SPS2 (middle), DIT1, DIT2 (mid-late) and SPS100 (late) genes as representative sporulation genes. We observed that sub1∆/sub1∆cells have a significantly elevated expression of middle genes (SPS2 and SMK1) that followed normal induction kinetics i.e., 5 hours post transfer to sporulation medium. However, the expression levels or timing for other class of sporulation genes did not change in sub1∆strain as compared with the wild-type. In order to confirm these observations, we also studied the effects of over-expression of SUB1 from the GAL1 promoter by transforming the high copy plasmid. This was done in wild-type SK1 cells and the expression of sporulation genes were analyzed. We observed that expression of SMK1 and SPS2middle sporulation genes was reduced on over-expression of SUB1.We used the Sub1-TAP protein to assess if Sub1 directly regulates these genes by Chromatin immunoprecipitation assays. For these studies, we examined the recruitment of Sub1 to these loci through the time course of sporulation. In wild-type SK1 cells, Sub1 was to bound to middle sporulation genes and this was striking in cells at 5th hour post-induction of sporulation. These data establish that Sub1 directly associates with chromatin at these loci co-incident with the time points where expression levels of these changes is altered in cells lacking Sub1. Furthermore, to assess the role of Sub1 in other stress responses, such as pseudohyphae formation in response to nitrogen starvation, pheromone-induced agar invasion and secretory stress, we employed a genetic approach. Genetic interaction studies of SUB1 with RPB4, a subunit of RNA polymerase with functions in stress response and HOS2, a subunit of Set3 complex and a close homolog of mammalian HDAC3, reported to be involved in sporulation and secretory stress, were performed. Based on sporulation frequency and pseudohyphal formation in the double mutants we conclude that SUB1 is downstream of both these genes. Moreover, our results from assays of schmoo formation and pheromone-induced agar invasion suggest that SUB1 functionally interacts with HOS2. Study of domain architecture of Sub1 and homology to human PC4 Comparison of the S. cerevisiae Sub1 protein with its higher eukaryotic homologs showed that the N-terminal region of yeast Sub1 (32-105 aa) is highly conserved (Knauset al., 1996; Henry et al., 1996) with the 106-292 C -terminal amino acids being yeast-specific. We employed deletion analysis to generate partial Sub1 proteins and used them to understand the roles played by these domains in different phenotypes associated with Sub1. Our analysis of the localization of various Sub1-GFP fusion proteins shows that 146-172 aa in the C-terminal domain of Sub1 confers nuclear localization. Sporulation frequency analysis of the different domains of Sub1 suggests that both the N and C terminal domains are necessary for sporulation function of Sub1. The N terminal domain of yeast Sub1 shares homology with human PC4 and not surprisingly possesses ssDNA binding ability first attributed to human PC4 (Kaiser et al., 1995). In order to investigate whether the effects of SUB1 on kinetics of sporulation require its ssDNA binding function, we generated the sub1(Y66A) ssDNA binding mutant (Sikorskiet al., 2011) and over-expressed it in the S288c genetic background. We assessed sporulation efficiency of sub1∆/sub1∆cells over-expressing sub1(Y66A) mutant allele as compared to cells over-expressing wild-type SUB1. Interestingly, cells with over-expression of sub1(Y66A) have reduced sporulation efficiency that is equivalent to the levels achieved on over-expression of wild type SUB1. This data suggests that the ssDNA-binding ability of Sub1 is not important for its role in sporulation. Furthermore, we examined the ability of human PC4 to contribute to yeast sporulation process by complementation analysis. We observed that over-expression of PC4 complemented the phenotypes of sub1∆strain, suggesting that the function of Sub1/PC4 family is evolutionarily conserved. Studies on biochemical interactions of Sub1 with histone proteins Human PC4 is a chromatin-associated protein, present on metaphase chromosomes (Das et al., 2006). The short C-terminal domain of PC4(62-87 aa) interacts with core histones H3 and H2B in vitro and in vivo and this interaction mediates chromatin condensation. The homology between S. cerevisiaeSub1 (32-105 aa) and human PC4 (62-127 aa)is in the domain required for their DNA binding properties and coactivator functions, suggesting possible conservation in their interactions. We tested the interactions of yeast Sub1 with histone proteins by adopting both in vitro and in vivo interaction assays. We find recombinant Sub1 had strong interactions with rat and yeast histone H3in vitro. Moreover,Sub1 was found to interact with histone H2B, but not with H2A, in vivo, a binding specificity also shown by human PC4.Thus, we demonstrate conservation in the interaction of Sub1 with histone proteins.