Characterization of systemic genomic instability in budding yeast.

Conventional models of genome evolution are centered around the principle that mutations form independently of each other and build up slowly over time. We characterized the occurrence of bursts of genome-wide loss-of-heterozygosity (LOH) in Saccharomyces cerevisiae, providing support for an additional nonindependent and faster mode of mutation accumulation. We initially characterized a yeast clone isolated for carrying an LOH event at a specific chromosome site, and surprisingly found that it also carried multiple unselected rearrangements elsewhere in its genome. Whole-genome analysis of over 100 additional clones selected for carrying primary LOH tracts revealed that they too contained unselected structural alterations more often than control clones obtained without any selection. We also measured the rates of coincident LOH at two different chromosomes and found that double LOH formed at rates 14- to 150-fold higher than expected if the two underlying single LOH events occurred independently of each other. These results were consistent across different strain backgrounds and in mutants incapable of entering meiosis. Our results indicate that a subset of mitotic cells within a population can experience discrete episodes of systemic genomic instability, when the entire genome becomes vulnerable and multiple chromosomal alterations can form over a narrow time window. They are reminiscent of early reports from the classic yeast genetics literature, as well as recent studies in humans, both in cancer and genomic disorder contexts. The experimental model we describe provides a system to further dissect the fundamental biological processes responsible for punctuated bursts of structural genomic variation.

Systemic and rapid restructuring of the genome: a new perspective on punctuated equilibrium.

The rates and patterns by which cells acquire mutations profoundly shape their evolutionary trajectories and phenotypic potential. Conventional models maintain that mutations are acquired independently of one another over many successive generations. Yet, recent evidence suggests that cells can also experience mutagenic processes that drive rapid genome evolution. One such process manifests as punctuated bursts of genomic instability, in which multiple new mutations are acquired simultaneously during transient episodes of genomic instability. This mutational mode is reminiscent of the theory of punctuated equilibrium, proposed by Stephen Jay Gould and Niles Eldredge in 1972 to explain the burst-like appearance of new species in the fossil record. In this review, we survey the dominant and emerging theories of eukaryotic genome evolution with a particular focus on the growing body of work that substantiates the existence and importance of punctuated bursts of genomic instability. In addition, we summarize and discuss two recent studies from our own group, the results of which indicate that punctuated bursts systemic genomic instability (SGI) can rapidly reconfigure the structure of the diploid genome of Saccharomyces cerevisiae.

Saccharomyces spores are born prepolarized to outgrow away from spore-spore connections and penetrate the ascus wall.

How nonspore haploid Saccharomyces cells choose sites of budding and polarize towards pheromone signals in order to mate has been a subject of intense study. Unlike nonspore haploids, sibling spores produced via meiosis and sporulation by a diploid cell are physically interconnected and encased in a sac derived from the old cell wall of the diploid, called the ascus. Nonspore haploids bud adjacent to previous sites of budding, relying on stable cortical landmarks laid down during prior divisions, but because spore membranes are made de novo, it was assumed that, as is known for fission yeast, Saccharomyces spores break symmetry and polarize at random locations. Here, we show that this assumption is incorrect: Saccharomyces cerevisiae spores are born prepolarized to outgrow, prior to budding or mating, away from interspore bridges. Consequently, when spores bud within an intact ascus, their buds locally penetrate the ascus wall, and when they mate, the resulting zygotes adopt a unique morphology reflective of repolarization towards pheromone. Long-lived cortical foci containing the septin Cdc10 mark polarity sites, but the canonical bud site selection programme is dispensable for spore polarity, thus the origin and molecular composition of these landmarks remain unknown. These findings demand further investigation of previously overlooked mechanisms of polarity establishment and local cell wall digestion and highlight how a key step in the Saccharomyces life cycle has been historically neglected.

Off-target effects of the septin drug forchlorfenuron on nonplant eukaryotes.

The septins are a family of GTP-binding proteins that form cytoskeletal filaments. Septins are highly conserved and evolutionarily ancient but are absent from land plants. The synthetic plant cytokinin forchlorfenuron (FCF) was shown previously to inhibit budding yeast cell division and induce ectopic septin structures (M. Iwase, S. Okada, T. Oguchi, and A. Toh-e, Genes Genet. Syst. 79:199-206, 2004, http://dx.doi.org/10.1266/ggs.79.199). Subsequent studies in a wide range of eukaryotes have concluded that FCF exclusively inhibits septin function, yet the mechanism of FCF action in nonplant cells remains poorly understood. Here, we report that the cellular effects of FCF are far more complex than previously described. The reported growth arrest of budding yeast cells treated with 1 mM FCF partly reflects sensitization caused by a bud4 mutation present in the W303 strain background. In wild-type (BUD4(+)) budding yeast, growth was inhibited at FCF concentrations that had no detectable effect on septin structure or function. Moreover, FCF severely inhibited the proliferation of fission yeast cells, in which septin function is nonessential. FCF induced fragmentation of budding yeast mitochondrial reticula and the loss of mitochondrial membrane potential. Mitochondria also fragmented in cultured mammalian cells treated with concentrations of FCF that previously were assumed to target septins only. Finally, FCF potently inhibited ciliation and motility and induced mitochondrial disorganization in Tetrahymena thermophila without apparent alterations in septin structure. None of these effects was consistent with the inhibition of septin function. Our findings point to nonseptin targets as major concerns when using FCF.

Bursts of Genomic Instability Potentiate Phenotypic and Genomic Diversification in Saccharomyces cerevisiae.

How microbial cells leverage their phenotypic potential to survive in a changing environment is a complex biological problem, with important implications for pathogenesis and species evolution. Stochastic phenotype switching, a particularly fascinating adaptive approach observed in numerous species across the tree of life, introduces phenotypic diversity into a population through mechanisms which have remained difficult to define. Here we describe our investigations into the mechanistic basis of colony morphology phenotype switching which occurs in populations of a pathogenic isolate of Saccharomyces cerevisiae, YJM311. We observed that clonal populations of YJM311 cells produce variant colonies that display altered morphologies and, using whole genome sequence analysis, discovered that these variant clones harbored an exceptional collection of karyotypes newly altered by de novo structural genomic variations (SVs). Overall, our analyses indicate that copy number alterations, more often than changes in allelic identity, provide the causative basis of this phenotypic variation. Individual variants carried between 1 and 16 de novo copy number variations, most of which were whole chromosomal aneuploidies. Notably, we found that the inherent stability of the diploid YJM311 genome is comparable to that of domesticated laboratory strains, indicating that the collections of SVs harbored by variant clones did not arise by a chronic chromosomal instability (CIN) mechanism. Rather, our data indicate that these variant clones acquired such complex karyotypic configurations simultaneously, during stochastic and transient episodes of punctuated systemic genomic instability (PSGI). Surprisingly, we found that the majority of these highly altered variant karyotypes were propagated with perfect fidelity in long-term passaging experiments, demonstrating that high aneuploidy burdens can often be conducive with prolonged genomic integrity. Together, our results demonstrate that colony morphology switching in YJM311 is driven by a stochastic process in which genome stability and plasticity are integrally coupled to phenotypic heterogeneity. Consequently, this system simultaneously introduces both phenotypic and genomic variation into a population of cells, which can, in turn perpetuate population diversity for many generations thereafter.

Genomic characterization of a wild diploid isolate of Saccharomyces cerevisiae reveals an extensive and dynamic landscape of structural variation.

The budding yeast Saccharomyces cerevisiae has been extensively characterized for many decades and is a crucial resource for the study of numerous facets of eukaryotic biology. Recent whole genome sequence analysis of over 1000 natural isolates of S. cerevisiae has provided critical insights into the evolutionary landscape of this species by revealing a population structure comprised of numerous genomically diverse lineages. These survey-level analyses have been largely devoid of structural genomic information, mainly because short-read sequencing is not suitable for detailed characterization of genomic architecture. Consequently, we still lack a complete perspective of the genomic variation that exists within this species. Single molecule long-read sequencing technologies, such as Oxford Nanopore and PacBio, provide sequencing-based approaches with which to rigorously define the structure of a genome, and have empowered yeast geneticists to explore this poorly described realm of eukaryotic genomics. Here, we present the comprehensive genomic structural analysis of a wild diploid isolate of S. cerevisiae, YJM311. We used long-read sequence analysis to construct a haplotype-phased, telomere-to-telomere length assembly of the YJM311 genome and characterized the structural variations (SVs) therein. We discovered that the genome of YJM311 contains significant intragenomic structural variation, some of which imparts notable consequences to the genomic stability and developmental biology of the strain. Collectively, we outline a new methodology for creating accurate haplotype-phased genome assemblies and highlight how such genomic analyses can be used to define the structural architectures of natural S. cerevisiae isolates. It is our hope that continued structural characterization of S. cerevisiae genomes, such as we have reported here for YJM311, will comprehensively advance our understanding of eukaryotic genome structure-function relationships, structural genomic diversity, and evolution.

Genome-Wide Analysis of Mitotic Recombination in Budding Yeast.

DNA break lesions pose a serious threat to the integrity of the genome. Eukaryotic cells can repair these lesions using the homologous recombination pathway that guides the repair reaction by using a homologous DNA template. The budding yeast Saccharomyces cerevisiae is an excellent model system with which to study this repair mechanism and the resulting patterns of genomic change resulting from it. In this chapter, we describe an approach that utilizes whole-genome sequencing data to support the analysis of tracts of loss-of-heterozygosity (LOH) that can arise from mitotic recombination in the context of the entire diploid yeast genome. The workflow and the discussion in this chapter are intended to enable classically trained molecular biologists and geneticists with limited experience in computational methods to conceptually understand and execute the steps of genome-wide LOH analysis as well as to adapt and apply them to their own specific studies and experimental models.

Punctuated Aneuploidization of the Budding Yeast Genome.

Remarkably complex patterns of aneuploidy have been observed in the genomes of many eukaryotic cell types, ranging from brewing yeasts to tumor cells. Such aberrant karyotypes are generally thought to take shape progressively over many generations, but evidence also suggests that genomes may undergo faster modes of evolution. Here, we used diploid Saccharomyces cerevisiae cells to investigate the dynamics with which aneuploidies arise. We found that cells selected for the loss of a single chromosome often acquired additional unselected aneuploidies concomitantly. The degrees to which these genomes were altered fell along a spectrum, ranging from simple events affecting just a single chromosome, to systemic events involving many. The striking complexity of karyotypes arising from systemic events, combined with the high frequency at which we detected them, demonstrates that cells can rapidly achieve highly altered genomic configurations during temporally restricted episodes of genomic instability.

Effectors of the spindle assembly checkpoint are confined within the nucleus of Saccharomyces cerevisiae.

The spindle assembly checkpoint (SAC) prevents erroneous chromosome segregation by delaying mitotic progression when chromosomes are incorrectly attached to the mitotic spindle. This delay is mediated by mitotic checkpoint complexes (MCCs), which assemble at unattached kinetochores and repress the activity of the anaphase promoting complex/cyclosome (APC/C). The cellular localizations of MCCs are likely critical for proper SAC function, yet remain poorly defined. We recently demonstrated that in mammalian cells, in which the nuclear envelope disassembles during mitosis, MCCs diffuse throughout the spindle region and cytoplasm. Here, we employed an approach using binucleate yeast zygotes to examine the localization dynamics of SAC effectors required for MCC assembly and function in budding yeast, in which the nuclear envelope remains intact throughout mitosis. Our findings indicate that in yeast, MCCs are confined to the nuclear compartment and excluded from the cytoplasm during mitosis.

"Wait anaphase" signals are not confined to the mitotic spindle.

The spindle assembly checkpoint ensures the faithful inheritance of chromosomes by arresting mitotic progression in the presence of kinetochores that are not attached to spindle microtubules. This is achieved through inhibition of the anaphase-promoting complex/cyclosome by a kinetochore-derived "wait anaphase" signal known as the mitotic checkpoint complex. It remains unclear whether the localization and activity of these inhibitory complexes are restricted to the mitotic spindle compartment or are diffusible throughout the cytoplasm. Here we report that "wait anaphase" signals are indeed able to diffuse outside the confines of the mitotic spindle compartment. Using a cell fusion approach to generate multinucleate cells, we investigate the effects of checkpoint signals derived from one spindle compartment on a neighboring spindle compartment. We find that spindle compartments in close proximity wait for one another to align all chromosomes before entering anaphase synchronously. Synchrony is disrupted in cells with increased interspindle distances and cellular constrictions between spindle compartments. In addition, when mitotic cells are fused with interphase cells, "wait anaphase" signals are diluted, resulting in premature mitotic exit. Overall our studies reveal that anaphase inhibitors are diffusible and active outside the confines of the mitotic spindle from which they are derived.

Roles of septins in prospore membrane morphogenesis and spore wall assembly in Saccharomyces cerevisiae.

The highly conserved family of septin proteins has important functions in cytokinesis in mitotically proliferating cells. A different form of cytokinesis occurs during gametogenesis in Saccharomyces cerevisiae, in which four haploid meiotic products become encased by prospore membrane (PSMs) and specialized, stress-resistant spore walls. Septins are known to localize in a series of structures near the growing PSM, but previous studies noted only mild sporulation defects upon septin mutation. We report that directed PSM extension fails in many septin-mutant cells, and, for those that do succeed, walls are abnormal, leading to increased susceptibility to heating, freezing, and digestion by the Drosophila gut. Septin mutants mislocalize the leading-edge protein (LEP) complex required for normal PSM and wall biogenesis, and ectopic expression of the LEP protein Ssp1 perturbs mitotic septin localization and function, suggesting a functional interaction. Strikingly, extra copies of septin CDC10 rescue sporulation and LEP localization in cells lacking Sma1, a phospholipase D-associated protein dispensable for initiation of PSM assembly and PSM curvature but required for PSM extension. These findings point to key septin functions in directing efficient membrane and cell wall synthesis during budding yeast gametogenesis.

Kinetic partitioning during de novo septin filament assembly creates a critical G1 "window of opportunity" for mutant septin function.

Septin proteins form highly conserved cytoskeletal filaments composed of hetero-oligomers with strict subunit stoichiometry. Mutations within one hetero-oligomerization interface (the "G" interface) bias the mutant septin toward conformations that are incompatible with filament assembly, causing disease in humans and, in budding yeast cells, temperature-sensitive defects in cytokinesis. We previously found that, when the amount of other hetero-oligomerization partners is limiting, wild-type and G interface-mutant alleles of a given yeast septin "compete" along parallel but distinct folding pathways for occupancy of a limited number of positions within septin hetero-octamers. Here, we synthesize a mathematical model that outlines the requirements for this phenomenon: if a wild-type septin traverses a folding pathway that includes a single rate-limiting folding step, the acquisition by a mutant septin of additional slow folding steps creates an initially large disparity between wild-type and mutant in the cellular concentrations of oligomerization-competent monomers. When the 2 alleles are co-expressed, this kinetic disparity results in mutant exclusion from hetero-oligomers, even when the folded mutant monomer is oligomerization-competent. To test this model experimentally, we first visualize the kinetic delay in mutant oligomerization in living cells, and then narrow or widen the "window of opportunity" for mutant septin oligomerization by altering the length of the G1 phase of the yeast cell cycle, and observe the predicted exacerbation or suppression, respectively, of mutant cellular phenotypes. These findings reveal a fundamental kinetic principle governing in vivo assembly of multiprotein complexes, independent of the ability of the subunits to associate with each other.

Assembly, molecular organization, and membrane-binding properties of development-specific septins.

Septin complexes display remarkable plasticity in subunit composition, yet how a new subunit assembled into higher-order structures confers different functions is not fully understood. Here, this question is addressed in budding yeast, where during meiosis Spr3 and Spr28 replace the mitotic septin subunits Cdc12 and Cdc11 (and Shs1), respectively. In vitro, the sole stable complex that contains both meiosis-specific septins is a linear Spr28-Spr3-Cdc3-Cdc10-Cdc10-Cdc3-Spr3-Spr28 hetero-octamer. Only coexpressed Spr3 and Spr28 colocalize with Cdc3 and Cdc10 in mitotic cells, indicating that incorporation requires a Spr28-Spr3 protomer. Unlike their mitotic counterparts, Spr28-Spr3-capped rods are unable to form higher-order structures in solution but assemble to form long paired filaments on lipid monolayers containing phosphatidylinositol-4,5-bisphosphate, mimicking presence of this phosphoinositide in the prospore membrane. Spr28 and Spr3 fail to rescue the lethality of a cdc11Δ cdc12Δ mutant, and Cdc11 and Cdc12 fail to restore sporulation proficiency to spr3Δ/spr3Δ spr28Δ/spr28Δ diploids. Thus, specific meiotic and mitotic subunits endow septin complexes with functionally distinct properties.

A Short CEP135 Splice Isoform Controls Centriole Duplication.

Centriole duplication is coordinated such that a single round of duplication occurs during each cell cycle. Disruption of this synchrony causes defects including supernumerary centrosomes in cancer and perturbed ciliary signaling [1-5]. To preserve the normal number of centrioles, the level, localization, and post-translational modification of centriole proteins is regulated so that, when centriole protein expression and/or activity are increased, centrioles self-assemble. Assembly is initiated by the formation of the cartwheel structure that comprises the base of centrioles [6-11]. SAS-6 constitutes the cartwheel, and SAS-6 levels remain low until centriole assembly is initiated at S phase onset [3, 12, 13]. CEP135 physically links to SAS-6 near the site of microtubule nucleation and binds to CPAP for triplet microtubule formation [13, 14]. We identify two distinct protein isoforms of CEP135 that antagonize each other to modulate centriole duplication: full-length CEP135 (CEP135(full)) promotes new assembly, whereas a short isoform, CEP135(mini), represses it. CEP135(mini) represses centriole duplication by limiting the centriolar localization of CEP135(full) binding proteins (SAS-6 and CPAP) and the pericentriolar localization of γ-tubulin. The CEP135 isoforms exhibit distinct and complementary centrosomal localization during the cell cycle. CEP135(mini) protein decreases from centrosomes upon anaphase onset. We suggest that the decrease in CEP135(mini) from centrosomes promotes centriole assembly. The repression of centriole duplication by a splice isoform of a protein that normally promotes it serves as a novel mechanism to limit centriole duplication.

An Inducible TGF-ß2-TGFßR Pathway Modulates the Sensitivity of HNSCC Cells to Tyrosine Kinase Inhibitors Targeting Dominant Receptor Tyrosine Kinases.

The epidermal growth factor receptor (EGFR) is overexpressed in approximately 90% of head and neck squamous cell carcinomas (HNSCC), and molecularly targeted therapy against the EGFR with the monoclonal antibody cetuximab modestly increases overall survival in head and neck cancer patients. We hypothesize that co-signaling through additional pathways limits the efficacy of cetuximab and EGFR-specific tyrosine kinase inhibitors (TKIs) in the clinical treatment of HNSCC. Analysis of gene expression changes in HNSCC cell lines treated 4 days with TKIs targeting EGFR and/or fibroblast growth factor receptors (FGFRs) identified transforming growth factor beta 2 (TGF-ß2) induction in the three cell lines tested. Measurement of TGF-ß2 mRNA validated this observation and extended it to additional cell lines. Moreover, TGF-ß2 mRNA was increased in primary patient HNSCC xenografts treated for 4 weeks with cetuximab, demonstrating in vivo relevance of these findings. Functional genomics analyses with shRNA libraries identified TGF-ß2 and TGF-ß receptors (TGFßRs) as synthetic lethal genes in the context of TKI treatment. Further, direct RNAi-mediated silencing of TGF-ß2 inhibited cell growth, both alone and in combination with TKIs. Also, a pharmacological TGFßRI inhibitor similarly inhibited basal growth and enhanced TKI efficacy. In summary, the studies support a TGF-ß2-TGFßR pathway as a TKI-inducible growth pathway in HNSCC that limits efficacy of EGFR-specific inhibitors.

Rapidly acquired resistance to EGFR tyrosine kinase inhibitors in NSCLC cell lines through de-repression of FGFR2 and FGFR3 expression.

Despite initial and sometimes dramatic responses of specific NSCLC tumors to EGFR TKIs, nearly all will develop resistance and relapse. Gene expression analysis of NSCLC cell lines treated with the EGFR TKI, gefitinib, revealed increased levels of FGFR2 and FGFR3 mRNA. Analysis of gefitinib action on a larger panel of NSCLC cell lines verified that FGFR2 and FGFR3 expression is increased at the mRNA and protein level in NSCLC cell lines in which the EGFR is dominant for growth signaling, but not in cell lines where EGFR signaling is absent. A luciferase reporter containing 2.5 kilobases of fgfr2 5' flanking sequence was activated after gefitinib treatment, indicating transcriptional regulation as a contributing mechanism controlling increased FGFR2 expression. Induction of FGFR2 and FGFR3 protein as well as fgfr2-luc activity was also observed with Erbitux, an EGFR-specific monoclonal antibody. Moreover, inhibitors of c-Src and MEK stimulated fgfr2-luc activity to a similar degree as gefitinib, suggesting that these pathways may mediate EGFR-dependent repression of FGFR2 and FGFR3. Importantly, our studies demonstrate that EGFR TKI-induced FGFR2 and FGFR3 are capable of mediating FGF2 and FGF7 stimulated ERK activation as well as FGF-stimulated transformed growth in the setting of EGFR TKIs. In conclusion, this study highlights EGFR TKI-induced FGFR2 and FGFR3 signaling as a novel and rapid mechanism of acquired resistance to EGFR TKIs and suggests that treatment of NSCLC patients with combinations of EGFR and FGFR specific TKIs may be a strategy to enhance efficacy of single EGFR inhibitors.