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Item Open Access A Role for Gic1 and Gic2 in Promoting Cdc42 Polarization(2018) Daniels, Christine NicoleThe Rho GTPase Cdc42 is a master regulator of cell polarity that orchestrates reorganization of the cytoskeleton. During polarity establishment, active GTP-Cdc42 accumulates at a part of the cell cortex that becomes the front of the cell. Localized GTP-Cdc42 orients the cytoskeleton through a set of “effector” proteins that bind specifically to GTP-Cdc42 and not GDP-Cdc42. A family of Cdc42 effectors, called GICs in yeast and BORGs in mammals, have been implicated in regulation of both the actin cytoskeleton and the septin cytoskeleton. Yeast cells lacking both Gic1 and Gic2 are able to polarize and grow at low temperatures, but many mutant cells fail to polarize the cytoskeleton at high temperature. This led to the conclusion that GICs communicate between Cdc42 and different cytoskeletal elements.
To better characterize the role of GIC proteins in yeast, we utilized time-lapse fluorescent microscopy to examine morphogenetic events in living single cells. Surprisingly, we found that not only the cytoskeleton but also Cdc42 itself failed to polarize in many gic1 gic2 mutant cells at high temperature. This observation indicates that GICs may act upstream of polarization rather than downstream.
Polarization of Cdc42 is triggered by cell-cycle progression, and in particular by G1 Cyclin-dependent kinase (CDK) activity. Using a live-cell reporter for G1 CDK activation, we found that cells lacking GICs were not defective in CDK activation, but showed a specific defect in polarization downstream of the CDK. Previous work had implicated the scaffold protein Bem1 in a positive feedback loop important for polarization. Cells lacking GICs failed to polarize Bem1 as well as Cdc42 at high temperature. Future work will be directed at understanding how GICs contribute to polarity establishment. Because many of the mechanisms and proteins involved in polarization are highly conserved, we anticipate our findings will help inform how this process regulated in higher eukaryotes.
Item Open Access Cell Polarity Establishment in the Budding Yeast Saccharomyces Cerevisiae(2009) Howell, AudreyEstablishing an axis of cell polarity is central to cell motility, tissue morphogenesis, and cell proliferation. A highly conserved group of polarity regulators is responsible for organizing a wide variety of polarized morphologies. One of the most widely expressed polarity regulators is the Rho-type GTPase Cdc42. In response to cell cycle cues the budding yeast Saccharomyces cerevisiae polarizes Cdc42p to a discrete site on the cell periphery. GTP-Cdc42p recruits a number of effectors that aid in the organization of a polarized actin cytoskeleton. The polarized actin cytoskeleton acts as tracks to facilitate the delivery of the secretory vesicles that will grow the bud, an essential process for an organism that proliferates by budding. We have employed treatment with the actin depolymerizing drugs Latrunculin A and B as well as high-speed timelapse microscopy of fluorescently labeled polarity proteins to characterize the assembly of the incipient bud site.
Often, ensuring that only a single axis of polarity is established is as important as generating asymmetry in the cell. Even in the absence of positional cues dictating the direction of polarization, many cells are still able to self-organize and establish one, and only one, polarity axis through a process termed symmetry breaking. Symmetry breaking is thought to employ positive feedback to amplify stochastic fluctuations in protein concentration into a larger asymmetry. To test whether singularity could be guaranteed by the amplification mechanism we re-wired yeast to employ a synthetic positive feedback mechanism. The re-wired cells could establish polarity, however they occasionally made two buds simultaneously, suggesting that singularity is guaranteed by the amplification mechanism.
Item Open Access Characterization of the Association of mRNA Export Factor Yra1 with the C-terminal Domain of RNA Polymerase II in vivo and in vitro(2011) MacKellar, AprilThe unique C-terminal domain (CTD) of RNA polymerase II (RNAPII), composed of tandem heptad repeats of the consensus sequence YSPTSPS, is subject to differential phosphorylation throughout the transcription cycle. Several RNA processing factors have been shown to bind the appropriately phosphorylated CTD, and this facilitates their localization to nascent pre-mRNA during transcription. In Saccharomyces cerevisiae, the mRNA export protein Yra1 (ALY/REF in metazoa) has been shown to cotranscriptionally associate with mRNA and is thought to deliver it to the nuclear pore complex for export to the cytoplasm. Based on a previous proteomics screen, I hypothesized that Yra1 is a bona fide phosphoCTD associated protein (PCAP) and that this interaction is responsible for the pattern of Yra1 cotranscriptional association observed in vivo. Using in vitro binding assays, I show that Yra1 directly binds the hyperphosphorylated form of the CTD characteristic of elongating RNAPII. Using truncations of Yra1, I determined that its phosphoCTD-interacting domain (PCID) resides in the segment comprising amino acids 18-184, which, interestingly, also contains the RNA Recognition Motif (RRM) (residues 77-184). Using UV crosslinking, I found that the RRM alone can bind RNA, although a larger protein segment, extending to the C-terminus (aa 77-226), displays stronger RNA binding activity. Even though the RRM is implicated in both RNA and CTD binding, certain RRM point mutations separate these two functions: thus, mutations that produce defects in RNA binding do not affect CTD binding. Both functions are important in vivo, in that RNA binding-defective or CTD binding-defective versions of Yra1 engender growth and mRNA export defects. I also report the construction and characterization of a useful new temperature sensitive YRA1 allele (R107AF126A). Finally, using chromatin immunoprecipitation, I demonstrate that removing the N-terminal 76 amino acids of Yra1 (all of the PCID up to the RRM) results in a 10-fold decrease in Yra1 recruitment to genes during elongation. These results indicate that the PCTD is likely involved directly in cotranscriptional recruitment of Yra1 to active genes.
Item Open Access Defining Roles for Cyclin Dependent Kinases and a Transcriptional Oscillator in the Organization of Cell-Cycle Events(2009) Simmons Kovacs, Laura AnneThe cell cycle is a series of ordered events that culminates in a single cell dividing into two daughter cells. These events must be properly coordinated to ensure the faithful passage of genetic material. How cell cycle events are carried out accurately remains a fundamental question in cell biology. In this dissertation, I investigate mechanisms orchestrating cell-cycle events in the yeast, Saccharomyces cerevisiae.
Cyclin dependent kinase (CDK) activity is thought to both form the fundamental cell-cycle oscillator and act as an effector of that oscillator, regulating cell-cycle events. By measuring transcript dynamics over time in cells lacking all CDK activity, I show that transcriptional oscillations are not dependent on CDK activity. This data indicates that CDKs do not form the underlying cell-cycle oscillator. I propose a model in which a transcription factor network rather than CDK activity forms the cell-cycle oscillator. In this model, CDKs are activated by the periodic transcription of cyclin genes and feedback on the network increasing the robustness of network oscillations in addition to regulating cell-cycle events.
I also investigate CDK-dependent and -independent mechanism regulating the duplication of the yeast centrosome, the spindle pole body (SPB). It is critical for the formation of a bipolar spindle in mitosis that the SPB duplicates once and only once per cell cycle. Through a combination of genetic and microscopic techniques I show that three distinct mechanisms regulate SPB duplication, ensuring its restriction to once per cell cycle.
Together, the data presented in this dissertation support a model in which CDKs, periodic transcription, and a TF-network oscillator are all important cell-cycle regulatory mechanisms that collaborate to regulate the intricate collection of events that constitute the cell cycle.
Item Open Access Experimentally informed bottom-up model of yeast polarity suggests how single cells respond to chemical gradients(2021) Ghose, DebrajHow do single cells—like neutrophils, amoebae, neurons, yeast, etc.—grow or move in a directed fashion in response to spatial chemical gradients? To address this question, we used the mating response in the budding yeast, Saccharomyces cerevisiae, as a biological model. To mate, pairs of yeast cells orient their cell fronts toward each other and fuse. Each cell relies on a pheromone gradient established by its partner to orient correctly. The ability for cells to resolve gradients is striking, because each cell is only ~5 μm wide and is thought to be operating in complex and noisy environments. Interestingly, mating pairs of cells often start out not facing each other. When this happens, the front of each cell—defined by a patch of cortical polarity proteins—undergoes a series of erratic and random movements along the cell cortex till it ‘finds’ the mating partner’s patch. We sought to understand how polarity patches in misaligned cells find each other. To this end, we first characterized patch movement in cells by the distribution of their step-lengths and turning angles and analyzed a bottom-up model of the polarity patch’s dynamics. The final version of our model combines 11 reaction-diffusion equations representing polarity protein dynamics with a stochastic module representing vesicle trafficking on a plane with periodic boundary conditions. We found that the model could not quantitatively reproduce step-length and turning angle distributions, which suggested that some mechanisms driving patch movement may not be present in the model. Incorpo-rating biologically inspired features into the model—such as focused vesicle delivery, sudden fluctuations in vesicle delivery rates, and the presence of polarity inhibitors on vesicles—allowed us to quantitatively match the in vivo polarity patch’s behavior. We then introduced a pathway, which connects pheromone sensing to polarity, to see how the model behaved when exposed to pheromone gradients. Concurrently, we analyzed the behavior of fluores-cently labeled polarity patches in mating pairs of cells. We discovered that the ~1 μm wide patch could (remarkably) sense and bias its movement up pheromone gradients, a result corroborated by our model. Further analysis of the model revealed that while the polarity patch tends to bias the location of a cluster of pheromone-sensing-receptors, the receptors can transform an external pheromone distribution into a peaked non-linear “polarity-activation” profile that “pulls” the patch. Stochastic perturbations cause the patch to “ping-pong” around the activation-profile. In a gradient of pheromone, this ping-ponging be-comes biased, leading to net patch movement up the gradient. We speculate that such a mechanism could be used by single cells with mobile fronts to track chemical gradients.
Item Open Access Gene Duplication and the Evolution of Silenced Chromatin in Yeasts(2010) Hickman, Meleah A.In Saccharomyces cerevisiae, proper maintenance of haploid cell identity requires the SIR complex to mediate the silenced chromatin found at the cryptic mating-type loci, HML and HMR. This complex consists of Sir2, a histone deacetylase and the histone binding proteins Sir3 and Sir4. Interestingly, both Sir2 and Sir3 have paralogs from a genome duplication that occurred after the divergence of Saccharomyces and Kluyveromyces species. The histone deacetylase HST1 is the paralog of SIR2 and works with the promoter-specific SUM1 complex to repress sporulation and alpha-specific genes. ORC1 is the paralog of SIR3 and is an essential subunit of the Origin Recognition Complex and also recruits SIR proteins to the HM loci. I have investigated the functions of these proteins in the non-duplicated species Kluyveromyces lactis and compared these functions to those found in S. cerevisiae.
I have shown that SIR2 and HST1 subfunctionalized post-duplication via the duplication, degeneration and complementation mechanism. In S. cerevisiae, Sir2 has retained the ability to function like Hst1 when in an hst1Δ strain. I have also shown, with a chimeric Sir2-Hst1 protein, that there are distinct specificity domains for Sir2 interaction with the SIR complex and Hst1 interaction with the SUM1 complex that have diverged between Sir2 and Hst1. Trans-species complementation assays show that the non-duplicated Sir2 from K. lactis can interact with both SIR and SUM1 complexes in S. cerevisiae.
Further analysis into the non-duplicated experimental system of K. lactis has revealed that deletion of KlSir2 de-represses the HM loci as well as sporulation and cell-type specific genes. A physical interaction between KlSir2 and the histone binding protein KlSir4 is conserved in K. lactis, and both proteins spread across the HML locus and associate with telomeres in a manner similar to S. cerevisiae. KlSir2 also physically interacts with the DNA-binding protein, KlSum1, to repress sporulation and cell-type specific genes in a promoter-specific manner and recruitment of KlSir2 to these loci is dependent on KlSum1. Surprisingly, deletion of KlSUM1 also de-represses HML and HMR, a phenotype not observed in S. cerevisiae. I show by chromatin immunoprecipitation that KlSum1 directly regulates the HM loci by spreading across these regions in a mechanism that is distinct from its role in repressing sporulation-specific genes. This result indicates that KlSum1 is a key regulator of not only meiotic, but also mating-type transcriptional programming.
The SIR3-ORC1 gene pair has previously been used as an example of neofunctionalization based on accelerated rates of evolution. However, my studies of KlOrc1 show it is distributed across HML and associates with Sir2 and Sir4 at telomeres, indicative of it having Sir3-like capabilities to spread across chromatin. This ability of KlOrc1 to spread is distinct from its functions with ORC, and is entirely dependent on its BAH domain. These findings demonstrate that prior to the genome duplication there was a silencing complex that contained both KlSir2 and KlOrc1. In addition to their functions at HML and the telomeres, KlOrc1 associates with replication origins and KlSir2 and KlSum1 work in complex to repress sporulation genes in a promoter-specific manner. The multiple functions of both KlOrc1 and KlSir2 in K. lactis indicate that after duplication, these properties were divided among paralogs and subsequently specialized to perform the functions that have been characterized in S. cerevisiae.
Item Open Access High-Resolution Mapping of Mitotic Recombination in Saccharomyces Cerevisiae(2012) St. Charles, Jordan AnneDouble-stranded DNA breaks are potentially lethal lesions that can be repaired in mitotic cells by either homologous recombination (HR) or non-homologous end- joining (NHEJ) pathways. In the HR pathway, the broken DNA molecule is repaired using either the sister chromatid or the homolog as a template. Mitotic recombination events involving the homolog often result in loss of heterozygosity (LOH) of markers located distal to the crossover. In humans that are heterozygous for a mutation in a tumor suppressor gene, mitotic recombination leading to LOH can be an early step in cancer development.
In my thesis research, I analyzed mitotic recombination in the yeast Saccharomyces cerevisiae using oligonucleotide-containing microarrays to detect LOH of single-nucleotide polymorphisms (SNPs). In analyzing cells treated with ionizing radiation, I performed the first whole-genome analysis of LOH events done in any organism (Chapter 2). I showed that irradiated cells had between two and three unselected LOH events. I also showed that crossovers were often associated with non- reciprocal exchanges of genetic information (gene conversion events) and that these conversion events were more complex than predicted by standard models of homologous recombination.
In Chapter 3, I describe my mapping of spontaneous crossovers in a 1.1 Mb region of yeast chromosome IV. This analysis is the first high-resolution mitotic recombination map of a substantial fraction (about 10%) of a eukaryotic genome. I demonstrated the existence of recombination "hotspots" and showed that some of these hotspots were homolog-specific. Two of the strongest hotspots were formed by closely- spaced inverted repeats of retrotransposons. I demonstrated that the hotspot activity was a consequence of a secondary DNA structure formed by these repeats. Additionally, the majority of spontaneous LOH events reflect DNA lesions induced in unreplicated chromosomes during G1 of the cell cycle, indicating that G1-initiated lesions threaten genome stability more than G2-initiated lesions.
In Chapter 4, I describe mitotic crossovers associated with DNA replication stress induced by hydroxyurea (HU) treatment. Surprisingly, most HU-induced crossovers had conversion tracts indicative of DNA lesions initiated in G1. Additionally, HU- induced recombination events were very significantly associated with solo delta elements, a 330 bp sequence that is repeated several hundred times in the yeast genome.
Item Open Access How Yeast Cells Find Their Mates(2019) Henderson, Nicholas TrubianoExtracellular chemical gradients provide signals that guide a broad spectrum of different cellular processes. By accurately sensing and responding to chemical gradients, immune cells can chase down invading pathogens, sperm cells can locate a distant egg, and growing axons can form connections in the developing nervous system. Haploid cells of the budding yeast Saccharomyces cerevisiae grow up gradients of pheromone in order to locate and fuse with nearby mating partners. Gradient sensing should be challenging for yeast, because they must detect a minute difference in concentration across their small cell bodies. Nevertheless, yeast cells can orient with remarkable accuracy in shallow pheromone gradients. Several mechanisms have been proposed to explain how yeast cells locate their partners, but it remains unclear whether, and to what degree each of the proposed mechanisms contributes.
We imaged fluorescent polarity probes in real time during mating events and found that cells located their partners in a multi-step process. First, cells placed a weak cluster of polarity proteins in approximately the correct location, despite a previously unappreciated challenge posed by asymmetrically distributed pheromone receptors. Cells were able to overcome receptor asymmetry by sensing the ratio, rather than the number, of active pheromone receptors. Second, the polarity cluster proceeded to move erratically around the cortex during an “indecisive phase.” We found evidence that cells switched to a local sensing mechanism during the indecisive phase, wherein cells used the mobile polarity cluster like a nose to search for areas where the local pheromone concentration was high. Third, the polarity cluster stabilized adjacent to a partner cell’s cluster and remained stationary until the partners met in the middle and fused.
Item Open Access Imaging Polarization in Budding Yeast.(Methods Mol Biol, 2016) McClure, Allison W; Wu, Chi-Fang; Johnson, Sam A; Lew, Daniel JWe describe methods for live-cell imaging of yeast cells that we have exploited to image yeast polarity establishment. As a rare event occurring on a fast time-scale, imaging polarization involves a trade-off between spatiotemporal resolution and long-term imaging without excessive phototoxicity. By synchronizing cells in a way that increases resistance to photodamage, we discovered unexpected aspects of polarization including transient intermediates with more than one polarity cluster, oscillatory clustering of polarity factors, and mobile "wandering" polarity sites.Item Open Access Investigations of Inositol Phosphate-Mediated Transcription(2012) Hatch, Ace JosephInositol phosphates (IPs) are eukaryotic signaling molecules that play important roles in a wide range of biological processes. IPs are required for embryonic development and patterning, insulin secretion, the regulation of telomere length, proper progression through the cell cycle, and the regulation of ion channels. This work uses the yeast Saccharomyces cerevisiae as a model system for investigating the functions of IPs and focuses on the transcriptional regulation of the gene encoding the secreted mating pheromone MFα2 by the IP kinase Ipk2 (also called Arg82, ArgR3, and IPMK). This work shows that Ipk2 has both kinase-dependent and kinase-independent functions in regulating the transcription of MFα2. Transcription of MFα2 is also dependent upon the integrity of an Mcm1-binding site in its promoter. This is the first description of a role for this binding site in the transcription of MFα2.
In vivo and in vitro screening approaches to identify additional factors associated with MFα2 expression or with IP biology generally are also described. These unbiased approaches provide some valuable insight for further investigations.
Item Open Access Mechanisms for Controlling Cell Polarity in Yeast(2022) Moran, Kyle DonovanCell polarity is critical for essential functions in many types of cells. Rho-family GTPases are master regulators of cell polarity. Thus, mechanisms for controlling the activity of Rho GTPases are of both academic interest and practical concern. While much has been discovered about regulation of Rho GTPase activity by partners like GEFs, GAPs, and GDIs, there are still many things which remain unclear about how their behavior is enforced in cells of various kinds.
The budding yeast, Saccharomyces cerevisiae, has long been a model for studying cell polarity. Its Rho GTPase Cdc42 is responsible for defining a single polarity site for the purpose of either mating with a single partner or making a single bud. Other types of yeasts, however, can generate multiple polarity sites utilizing the same core polarity machinery. What are the rules which allow for such differences in behavior while using a similar set of proteins? We highlight key design principles established in mathematical models for Rho GTPase polarity machineries and show that they apply in budding yeast cells: strains featuring specific genetic perturbations which increase the total amount of polarity proteins can go from making one bud, to making multiple buds.
Bud emergence in yeast is enabled by cell cycle activity in G1. It is known that bud emergence also requires cytoskeletal changes which are orchestrated by Cdc42 and its effectors. How are these changes coordinated with cell cycle progression? It seems likely that G1 CDK activity regulates many aspects of Cdc42 polarization. We use live-cell fluorescence microscopy to reveal one such avenue whereby input from the cell cycle is required for many Cdc42 effector proteins to localize to sites with active Cdc42, thus restricting bud formation until the time is right.
Item Open Access Mechanisms of Gradient Tracking During Yeast Mating(2012) Johnson, Jayme MMany cells are remarkably proficient at tracking even shallow chemical gradients, despite tiny differences in receptor occupancy across the cell. Stochastic receptor-ligand interactions introduce considerable noise in instantaneous receptor occupancy, so it is thought that spatial information must be integrated over time to allow noise filtering. The mechanism of temporal integration is unknown. We used the mating response of the budding yeast, Saccharomyces cerevisiae, as a model to study eukaryotic gradient tracking.
During mating, yeast cells polarize and grow up a gradient of pheromone to find and fuse with opposite-sex partners. Exposure to pheromone causes polarity regulators to cluster into a tight "patch" at the cortex, directing growth toward that site. Timelapse microscopy of fluorescently-labeled polarity proteins revealed that the patch wandered around the cortex during gradient tracking. Mathematical modeling and genetic analysis suggested that fusion of vesicles near the polarization site could perturb the polarity patch and promote wandering. Wandering is decreased due to global effects from pheromone signaling as well as interactions between receptor-activated Gβ and the exchange factor for the polarity regulator Cdc42. We found that artificially stabilizing patch wandering impaired accurate gradient tracking.
We suggest that ongoing polarized vesicle traffic causes patch wandering, which is locally reduced by pheromone-bound receptors. Thus, over time, spatial information from the pheromone gradient biases the random wandering of the polarity patch so that growth occurs predominantly up-gradient. Such temporal integration may enable sorting the low signal from stochastic noise when tracking shallow gradients.
Item Open Access Molecular Characterization of Mitotic Homologous Recombination Outcomes in Saccharomyces cerevisiae(2017) Hum, Yee FangMitotic homologous recombination (HR) is vital for accurate repair of DNA strand breaks caused by endogenous and exogenous sources. However, this high-fidelity repair pathway also can lead to genome rearrangements when dispersed sequences are used for repair. During normal growth, spontaneous DNA strand breaks are presumably generated during DNA replication and transcription, and from the attack by endogenous agents such as reactive oxygen species (ROS). Though the exact nature of endogenous lesions that initiate HR is not well understood, double-strand breaks (DSBs) rather than single-strand breaks (SSBs) are thought to be the main culprit. Because spontaneous HR events can lead to development of human diseases and sporadic cancers, identifying the primary type of DNA strand breaks, either DSBs or SSBs, is central to understanding how genome instability arises. Using the yeast Saccharomyces cerevisiae as a model system, the focus of this thesis is to delineate early molecular steps (DNA end resection and synthesis) during mitotic DSB-induced HR events and to perform comparative analysis of DSB-induced and spontaneous HR repair outcomes. To this end, the first part of this thesis examined the relative contribution of DNA end resection and DNA synthesis in determining the DSB-associated repair outcomes, such as distributions of crossover and noncrossover outcomes, as well as the length of a key recombination intermediate, heteroduplex (hetDNA). The main conclusion from this work is that both end resection and DNA synthesis are required to obtain normal DNA repair outcomes and hetDNA profiles. A unifying model is that decreased end resection reduces stability of strand invasion intermediates, limiting the extent of DNA synthesis and hence shortening hetDNA. The second and third parts of this work directly compared the molecular structures of HR outcomes associated with a defined DSB and with those arising spontaneously. Two different approaches were employed to systematically characterize the molecular structures of recombination intermediates in repair events. In the second part of this thesis, mapping of gene conversion events (nonreciprocal transfer of information that results from mismatch-repair activity) following allelic repair of a DSB revealed that DSB-induced HR events shared similar repair profiles with those of previously described spontaneous recombination events, confirming that DSBs are the main contributor to spontaneous HR. In the third part of this thesis, mapping of hetDNA in DSB-induced and spontaneous HR events in cells with normal and elevated ROS levels further confirmed that DSBs are the primary initiator of spontaneous HR. Mapping of hetDNA additionally revealed complexities within hetDNA associated with a defined DSB. Collectively, this work not only advances our knowledge of the fundamental molecular mechanisms of HR, but also provides in vivo experimental support for DSBs as the major physiological lesion that initiates spontaneous HR.
Item Open Access Negative Feedback and Competition in the Yeast Polarity Establishment Circuit(2013) Wu, ChiFangMany cells spontaneously establish a polarity axis even in the absence of directional cues, a process called symmetry breaking. A central question concerns how cells polarize towards one, and only one, randomly oriented "front". The conserved Rhotype GTPase Cdc42p is an essential factor for both directed and spontaneous polarization in various organisms, whose local activation is thought to define the cell's front. We previously proposed that in yeast cells, a small stochastic cluster of GTP-Cdc42p at a random site on the cortex can grow into a large, dominating cluster via a positive feedback loop involving the scaffold protein Bem1p. As stochastic Cdc42p clusters could presumably arise at many sites, why does only one site become the dominating "front"? We speculated that competition between growing clusters for limiting factors would lead to growth of a single winning "front" at the expense of the others. Utilizing time-lapse imaging with high spatiotemporal resolution, we now document initiation of multiple polarized clusters that competed rapidly to resolve a winning cluster. Such multicluster intermediates are observed in wild-type yeast cells with functional directional cues, but the locations where they are initiated are biased by the spatial cues. In addition, we detected an unexpected oscillatory polarization in a majority of the cells breaking symmetry, in which polarity factors initially concentrated very brightly and then dimmed in an oscillatory manner, dampening down to a final intermediate level after 2-3 peaks. Dampened oscillation suggests that the polarity circuit contains an in-built negative feedback loop. Mathematical modeling predicts that negative feedback would confer robustness to the polarity circuit and make the kinetics of competition between polarity factor clusters relatively insensitive to polarity factor concentration.
We are trying to understand how competition between clusters occurs. We find that the yeast guanine-nucleotide dissociation inhibitor (GDI), Rdi1p, is needed for rapid competition between clusters. In the absence of Rdi1p the initial clustering of polarity
factors is slowed, and competition is also much slower: in some cases cells still have two clusters at the time of bud emergence and they form two buds. We suggest that in the absence of Rdi1p, the clusters compete for a limiting pool of Cdc42p, and that slow
exchange of Cdc42p on and off the membrane in the absence of Rdi1p leads to slow competition.
Item Open Access Physical and Genetic Analysis of the CUP1 Tandem Array in the Yeast Saccharomyces cerevisiae(2016) Zhao, YingThe genomes of many strains of baker’s yeast, Saccharomyces cerevisiae, contain multiple repeats of the copper-binding protein Cup1. Cup1 is a member of the metallothionein family, and is found in a tandem array on chromosome VIII. In this thesis, I describe studies that characterized these tandem arrays and their mechanism of formation across diverse strains of yeast. I show that CUP1 arrays are an illuminating model system for observing recombination in eukaryotes, and describe insights derived from these observations.
In our first study, we analyzed 101 natural isolates of S. cerevisiae in order to examine the diversity of CUP1-containing repeats across different strains. We identified five distinct classes of repeats that contain CUP1. We also showed that some strains have only a single copy of CUP1. By comparing the sequences of all the strains, we were able to elucidate the mechanism of formation of the CUP1 tandem arrays, which involved unequal non-homologous recombination events starting from a strain that had only a single CUP1 gene. Our observation of CUP1 repeat formation allows more general insights about the formation of tandem repeats from single-copy genes in eukaryotes, which is one of the most important mechanisms by which organisms evolve.
In our second study, we delved deeper into our mechanistic investigations by measuring the relative rates of inter-homolog and intra-/inter-sister chromatid recombination in CUP1 tandem arrays. We used a diploid strain that is heterozygous both for insertion of a selectable marker (URA3) inside the tandem array, and also for markers at either end of the array. The intra-/inter-sister chromatid recombination rate turned out to be more than ten-fold greater than the inter-homolog rate. Moreover, we found that loss of the proteins Rad51 and Rad52, which are required for most inter-homolog recombination, did not greatly reduce recombination in the CUP1 tandem repeats. Additionally, we investigated the effects of elevated copper levels on the rate of each type of recombination at the CUP1 locus. Both types of recombination are increased at high concentrations of copper (as is known to be the case for CUP1 transcription). Furthermore, the inter-homolog recombination rate at the CUP1 locus is higher than the average over the genome during mitosis, but is lower than the average during meiosis.
The research described in Chapter 2 is published in 2014.
Item Open Access Regulation of Global Transcription Dynamics During Cell Division and Root Development(2009) Orlando, David AnthonyThe successful completion of many critical biological processes depends on the proper execution of complex spatial and temporal gene expression programs. With the advent of high-throughput microarray technology, it is now possible to measure the dynamics of these expression programs on a genome-wide level. In this thesis we present work focused on utilizing this technology, in combination with novel computational techniques, to examine the role of transcriptional regulatory mechanisms in controlling the complex gene expression programs underlying two fundamental biological processes---the cell cycle and the development and differentiation of an organ.
We generate a dataset describing the genomic expression program which occurs during the cell division cycle of Saccharomyces cerevisiae. By concurrently measuring the dynamics in both wild-type and mutant cells that do not express either S-phase or mitotic cyclins we quantify the relative contributions of cyclin-CDK complexes and transcriptional regulatory networks in the regulation the cell cell expression program. We show that CDKs are not the sole regulators of periodic transcription as contrary to previously accepted models; and we hypothesize an oscillating transcriptional regulatory network which could work independent of, or in tandem with, the CDK oscillator to control the cell cell expression program.
To understand the acquisition of cellular identity, we generate a nearly complete gene expression map of the Arabidopsis Thaliana root at the resolution of individual cell-types and developmental stages. An analysis of this data reveals a representative set of dominant expression patterns which are used to begin defining the spatiotemporal transcriptional programs that control development within the root.
Additionally, we develop computational tools that improve the interpretability and power of these data. We present CLOCCS, a model for the dynamics of population synchrony loss in time-series experiments. We demonstrate the utility of CLOCCS in integrating disparate datasets and present a CLOCCS based deconvolution of the cell-cycle expression data. A deconvolution method is also developed for the Arabidopsis dataset, increasing its resolution to cell-type/section subregion specificity. Finally, a method for identifying biological processes occurring on multiple timescales is presented and applied to both datasets.
It is through the combination of these new genome-wide expression studies and computational tools that we begin to elucidate the transcriptional regulatory mechanisms controlling fundamental biological processes.
Item Open Access Studies of Spontaneous Oxidative and Frameshift Mutagenesis in Saccharomyces cerevisiae(2010) Mudrak, Sarah VictoriaPreserving genome stability is critical to ensure the faithful transmission of intact genetic material through each cell division. One of the key components of this preservation is maintaining low levels of mutagenesis. Most mutations arise during replication of the genome, either as polymerase errors made when copying an undamaged DNA template or during the bypass of DNA lesions. Many different DNA repair proteins act both prior to and during replication to prevent the occurrence of these mutations. Although the mechanisms by which mutations occur and the various repair proteins that act to suppress mutagenesis are conserved throughout all species, they are best characterized in the yeast Saccharomyces cerevisiae. In this work, we have used this model system to study two types of spontaneous mutagenesis: oxidative mutagenesis and frameshift mutagenesis. In the first part of this work, we have examined mutagenesis that arises due to one of the most common oxidative lesions in the cell, 7,8-dihydro-8-oxoguanine or GO. When present during replication, these GO lesions generate characteristic transversion events that are accurately repaired by the mismatch repair pathway. We provide the first evidence that a second pathway involving the translesion synthesis polymerase Pol&eta acts independently of the mismatch repair pathway to suppress GO-associated mutagenesis. We have also examined how differences in replication timing during S phase contribute to variations in the rate of these mutations across the genome. In the second part of this work, we have examined how spontaneous frameshift mutations are generated during replication. While most frameshift mutations occur in regions of repetitive DNA, we have designed a system to examine frameshifts that occur in very short repeats (< 4 nucleotides) and noniterated sequences. We have examined the patterns of frameshifts at these sites and how the mismatch repair pathway acts to suppress these mutations. Together, the experiments presented here provide further insight into the different mechanisms that suppress and/or influence rates of oxidative mutagenesis and describe a system in which we have begun to characterize how frameshift mutations are generated at very short repeats and non-repetitive DNA.
Item Open Access The Evolution of the Deacetylase Sir2 in Yeast(2012) Froyd, Cara AnneGene duplication is an important evolutionary tool for fostering diversification and expanding gene families. However, while this concept is well understood and accepted in a theoretical capacity, the particular changes that lead to the functional diversification of gene duplicates are less well understood and documented. Additionally, little work has been done to understand how functions are gained or lost, which leads to the diversification of orthologous genes. The Sir2 family of NAD+-dependent deacetylases is an excellent gene family to study questions of duplication and diversification as it is ubiquitous throughout all kingdoms of life, and it has expanded through a number of gene duplications so that while most bacteria have a single sirtuin/species, mammals have seven sirtuins/species. Sirtuins also have a wide array of biological functions and targets, but some of these functions are conserved in eukaryotes.
In this study, Sir2 is used to investigate the principles behind gene duplication and functional diversification in a molecular context. Sir2 function is studied in multiple species of budding yeast, the model organism Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida lusitaniae using a combination of genetic, biochemical, and high-throughput methods. Sir2 and its paralog Hst1 from S. cerevisiae were used with their non-duplicated ortholog Sir2 from K. lactis to examine the type of molecular changes that occur after gene duplication and lead to subfunctionalization. Then Sir2 from the more divergent C. lusitaniae was used to study how functions are gained or lost.
To study the molecular mechanism of subfunctionalization in the duplicated deacetylases ScSir2 and ScHst1 with the non-duplicated KlSir2 used as a proxy for the ancestral state, we hypothesized that the basis for subfunctionalization in this case was in the interaction domains. ScSir2 and ScHst1 act in distinct complexes that target them to the genomic loci they regulate. KlSir2 interacts with the same complexes as both ScSir2 and ScHst1. Therefore, we first identified the minimal regions of ScSir2 and ScHst1 necessary for each to interact with its respective complex. Then we identified mutations in those interaction domains that eliminated those interactions. Those mutations were then tested in KlSir2 for their impact on its interactions with the same complexes. We found that the interaction domains in ScSir2 and ScHst1 were conserved in KlSir2, demonstrating that Sir2 and Hst1 subfunctionalized by acquiring complementary inactivating mutations in these interaction domains.
To understand better how Sir2 has gained or lost functions, we studied the Sir2 function in C. lusitaniae to serve as an intermediate between the fission yeast Schizosaccharomyces pombe Sir2, whose functions have been identified, and K. lactis and S. cerevisiae. Interestingly, ClSir2 was localized to the rDNA, which is also the case in S. pombe, K. lactis, and S. cerevisiae, but not at the telomeres, which is another locus at which Sir2 is found in other yeast. Additionally, ClSir2 was not found to have an impact on gene expression unlike Sir2 and Hst1 in other yeast where they repress transcription.
Item Open Access The Mechanism of Mitotic Recombination in Yeast(2010) Lee, Phoebe S.A mitotically dividing cell regularly experiences DNA damage including double-stranded DNA breaks (DSBs). Homologous mitotic recombination is an important mechanism for the repair of DSBs, but inappropriate repair of DNA breaks can lead to genome instability. Despite more than 70 years of research, the mechanism of mitotic recombination is still not understood. By genetic and physical studies in the yeast Saccharomyces cerevisiae, I investigated the mechanism of reciprocal mitotic crossovers. Since spontaneous mitotic recombination events are very infrequent, I used a diploid strain that allowed for selection of cells that had the recombinant chromosomes expected for a reciprocal crossover (RCO). The diploid was also heterozygous for many single-nucleotide polymorphisms, allowing the accurate mapping of the recombination events.
I mapped spontaneous crossovers to a resolution of about 4 kb in a 120 kb region of chromosome V. This analysis is the first large-scale mapping of mitotic events performed in any organism. One region of elevated recombination was detected (a "hotspot") and the region near the centromere of chromosome V had low levels of recombination ("coldspot"). This analysis also demonstrated the crossovers were often associated with the non-reciprocal transfer of information between homologous chromosomes; such events are termed "gene conversions" and have been characterized in detail in the products of meiotic recombination. The amount of DNA transferred during mitotic gene conversion events was much greater than that observed for meiotic conversions, 12 kb and 2 kb, respectively. In addition, about 40% of the conversion events had patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.
To confirm this unexpected conclusion, I examined the crossovers and gene conversion events induced by gamma irradiation in G1- and G2-arrested diploid yeast cells. The gene conversion patterns of G1-irradiated cells (but not G2-irradiated cells) mimic the conversion events associated with spontaneous reciprocal crossovers (RCOs), confirming my hypothesis that many spontaneous crossovers are initiated by a DSB on an unreplicated chromosome. In conclusion, my results have resulted in a new understanding of the properties of mitotic recombination within the context of cell cycle.