Diploid Budding Yeast Research Articles

Multi-signal regulation of the GSK-3β homolog Rim11 controls meiosis entry in budding yeast

Starvation in diploid budding yeast cells triggers a cell-fate program culminating in meiosis and spore formation. Transcriptional activation of early meiotic genes (EMGs) hinges on the master regulator Ime1, its DNA-binding partner Ume6, and GSK-3β kinase Rim11. Phosphorylation of Ume6 by Rim11 is required for EMG activation. We report here that Rim11 functions as the central signal integrator for controlling Ume6 phosphorylation and EMG transcription. In nutrient-rich conditions, PKA suppresses Rim11 levels, while TORC1 retains Rim11 in the cytoplasm. Inhibition of PKA and TORC1 induces Rim11 expression and nuclear localization. Remarkably, nuclear Rim11 is required, but not sufficient, for Rim11-dependent Ume6 phosphorylation. In addition, Ime1 is an anchor protein enabling Ume6 phosphorylation by Rim11. Subsequently, Ume6-Ime1 coactivator complexes form and induce EMG transcription. Our results demonstrate how various signaling inputs (PKA/TORC1/Ime1) converge through Rim11 to regulate EMG expression and meiosis initiation. We posit that the signaling-regulatory network elucidated here generates robustness in cell-fate control.

Mutagenicity in haploid yeast meiosis resulting from repair of DSBs by the sister chromatid.

Mutations in diploid budding yeast occur in meiosis at higher frequencies than in cells grown vegetatively. Such meiotic mutations are thought to result from the repair of double-strand breaks (DSBs) in meiosis, during the process of recombination. Here, we report studies of mutagenicity in haploid strains that may undergo meiosis due to the expression of both mating-type alleles, MATa and MATα. We measure the rate of mutagenicity in the reporter gene CAN1, and find it to be fivefold higher than in mitotic cells, as determined by fluctuation analysis. This enhanced meiotic mutagenicity is shown to depend on the presence of SPO11, the gene responsible for meiotic DSBs. Mutations in haploid meiosis must result from repair of the DSBs through interaction with the sister chromatid, rather than with non-sister chromatids as in diploids. Thus, mutations in diploid meiosis that are not ostensibly associated with recombination events can be explained by sister-chromatid repair. The spectrum of meiotic mutations revealed by Sanger sequencing is similar in haploid and in diploid meiosis. Compared to mitotic mutations in CAN1, long Indels are more frequent among meiotic mutations. Both, meiotic and mitotic mutations are more common at G/C sites than at A/T, in spite of an opposite bias in the target reporter gene. We conclude that sister-chromatid repair of DSBs is a major source of mutagenicity in meiosis.

Identification and in silico analysis of the origin recognition complex in the human fungal pathogen Candida albicans.

DNA replication in eukaryotes is initiated by the orchestrated assembly and association of initiator proteins (heterohexameric Origin Recognition Complex, ORC) on the replication origins. These functionally conserved proteins play significant roles in diverse cellular processes besides their central role in ignition of DNA replication at origins. Candida albicans, a major human fungal pathogen, is a diploid budding yeast that belongs to Ascomycota. However, C. albicans is significantly diverged from a well-studied model organism Saccharomyces cerevisiae, another ascomycete. The components of the DNA replication machinery in C. albicans remain largely uncharacterized. Identification of factors required for DNA replication is essential for understanding the evolution of the DNA replication machinery. We identified the putative ORC homologs in C. albicans and determined their relatedness with those of other eukaryotes including several yeast species. Our extensive in silico studies demonstrate that the domain architecture of CaORC proteins share similarities with the ORC proteins of S. cerevisiae. We dissect the domain organization of ORC (trans-acting factors) subunits that seem to associate with DNA replication origins in C. albicans. We present a model of the 3D structure of CaORC4 to gain further insights of this protein’s function.

Cis- and Trans-chromosomal Interactions Define Pericentric Boundaries in the Absence of Conventional Heterochromatin.

The diploid budding yeast Candida albicans harbors unique CENPA-rich 3- to 5-kb regions that form the centromere (CEN) core on each of its eight chromosomes. The epigenetic nature of these CENs does not permit the stabilization of a functional kinetochore on an exogenously introduced CEN plasmid. The flexible nature of such centromeric chromatin is exemplified by the reversible silencing of a transgene upon its integration into the CENPA-bound region. The lack of a conventional heterochromatin machinery and the absence of defined boundaries of CENPA chromatin makes the process of CEN specification in this organism elusive. Additionally, upon native CEN deletion, C. albicans can efficiently activate neocentromeres proximal to the native CEN locus, hinting at the importance of CEN-proximal regions. In this study, we examine this CEN-proximity effect and identify factors for CEN specification in C. albicans We exploit a counterselection assay to isolate cells that can silence a transgene when integrated into the CEN-flanking regions. We show that the frequency of reversible silencing of the transgene decreases from the central core of CEN7 to its peripheral regions. Using publicly available C. albicans high-throughput chromosome conformation capture data, we identify a 25-kb region centering on the CENPA-bound core that acts as CEN-flanking compact chromatin (CFCC). Cis- and trans-chromosomal interactions associated with the CFCC spatially segregates it from bulk chromatin. We further show that neocentromere activation on chromosome 7 occurs within this specified region. Hence, this study identifies a specialized CEN-proximal domain that specifies and restricts the centromeric activity to a unique region.

The protein expression landscape of mitosis and meiosis in diploid budding yeast

Saccharomyces cerevisiae is an established model organism for the molecular analysis of fundamental biological processes. The genomes of numerous strains have been sequenced, and the transcriptome and proteome ofmajor phases during the haploid and diploid yeast life cycle have been determined. However, much less is known about dynamic changes of the proteome when cells switch from mitotic growth to meiotic development. We report a quantitative protein profiling analysis of yeast cell division and differentiation based on mass spectrometry. Information about protein levels was integrated with strand-specific tiling array expression data. We identified a total of 2366 proteins in at least one condition, including 175 proteins showing a statistically significant>5-fold change across the sample set, and 136 proteins detectable in sporulating but not respiring cells. We correlate protein expression patterns with biological processes and molecular function by Gene Ontology term enrichment, chemoprofiling, transcription interference and the formation of double stranded RNAs by overlapping sense/antisense transcripts. Our work provides initial quantitative insight into protein expression in diploid respiring and differentiating yeast cells. Critically, it associates developmentally regulated induction of antisense long noncoding RNAs and double stranded RNAs with fluctuating protein concentrations during growth and development. Biological significanceThis integrated genomics analysis helps better understand how the transcriptome and the proteome correlate in diploid yeast cells undergoing mitotic growth in the presence of acetate (respiration) versus meiotic differentiation (Meiosis I and II). The study (i) provides quantitative expression data for 2366 proteins and their cognate mRNAs in at least one sample, (ii) shows strongly fluctuating protein levels during growth and differentiation for 175 cases, and (iii) identifies 136 proteins absent in mitotic but present in meiotic yeast cells. We have integrated protein profiling data using mass spectrometry with tiling array RNA profiling data and information on double-stranded RNAs (dsRNAs) by overlapping sense/antisense transcripts from an RNA-Sequencing experiment. This work therefore provides quantitative insight into protein expression during cell division and development and associates changing protein levels with developmental stage specific induction of antisense transcripts and the formation of dsRNAs.

Similar environments but diverse fates: Responses of budding yeast to nutrient deprivation.

Diploid budding yeast (Saccharomyces cerevisiae) can adopt one of several alternative differentiation fates in response to nutrient limitation, and each of these fates provides distinct biological functions. When different strain backgrounds are taken into account, these various fates occur in response to similar environmental cues, are regulated by the same signal transduction pathways, and share many of the same master regulators. I propose that the relationships between fate choice, environmental cues and signaling pathways are not Boolean, but involve graded levels of signals, pathway activation and master-regulator activity. In the absence of large differences between environmental cues, small differences in the concentration of cues may be reinforced by cell-to-cell signals. These signals are particularly essential for fate determination within communities, such as colonies and biofilms, where fate choice varies dramatically from one region of the community to another. The lack of Boolean relationships between cues, signaling pathways, master regulators and cell fates may allow yeast communities to respond appropriately to the wide range of environments they encounter in nature.

Interallelic interaction and gene regulation in budding yeast

InDrosophila, homologous chromosome pairing leads to "transvection," in which the enhancer of a gene can regulate the allelic transcription intrans.Interallelic interactions were also observed in vegetative diploid budding yeast, but their functional significance is unknown. Here, we show that aGAL1reporter can interact with its homologous allele and affect its expression. By ectopically inserting two allelic reporters, one driven by wild-typeGAL1promoter (WTGAL1pr) and the other by a mutant promoter with delayed response to galactose induction, we found that the two reporters physically associate, and the WTGAL1prtriggers synchronized firing of the defective promoter and accelerates its activation without affecting its steady-state expression level. This interaction and the transregulatory effect disappear when the same reporters are located at nonallelic sites. We further demonstrated that the activator Gal4 is essential for the interallelic interaction, and the transregulation requires fully activated WTGAL1prtranscription. The mechanism of this phenomenon was further discussed. Taken together, our data revealed the existence of interallelic gene regulation in yeast, which serves as a starting point for understanding long-distance gene regulation in this genetically tractable system.

Integrated RNA- and protein profiling of fermentation and respiration in diploid budding yeast provides insight into nutrient control of cell growth and development.

Our integrated genomics study provides insight into the interplay between the transcriptome and the proteome in diploid yeast cells undergoing vegetative growth in the presence of glucose (fermentation) or acetate (respiration). Furthermore, it reveals novel target genes involved in these processes for Ume6, the DNA binding subunit of the conserved histone deacetylase Rpd3 and the co-repressor Sin3. We have combined data from an RNA profiling experiment using tiling arrays that cover the entire yeast genome, and a large-scale protein detection analysis based on mass spectrometry in diploid MATa/α cells. This distinguishes our study from most others in the field-which investigate haploid yeast strains-because only diploid cells can undergo meiotic development in the simultaneous absence of a non-fermentable carbon source and nitrogen. Indeed, we report molecular clues how respiration of acetate might prime diploid cells for efficient spore formation, a phenomenon that is well known but poorly understood.

Correlations in Chromatin Movement in Diploid Yeast Revealed by Two-Color Three-Dimensional Single-Particle Tracking using the Double-Helix Point Spread Function (DH-PSF) Microscope

It is known that gene regulation in eukaryotes is intimately tied to the three-dimensional (3D) organization of chromatin within the nucleus, and the exact structure and dynamics of this organization is a current topic of intense investigation. In order to examine whether the activation of two alleles in a diploid genome is coordinated we performed fast (10 Hz), 3D, simultaneous tracking of the two alleles of the GAL locus in diploid budding yeast cells. We did so by labeling one copy of the locus with LacO/LacI-GFP and the other with TetO/TetR-mCherry and recording the resulting fluorescence images in parallel using the DH-PSF microscope, which enabled precise sub-diffraction 3D localization in each channel ( = [14,13,27] nm for GFP and [25,29,46] nm for mCherry). The simultaneously recorded tracks were then registered to within sub-diffraction distances. We then examined the statistics of allele velocity cross-correlation and colocalization in both the transcriptionally active (cells grown in galactose) and repressed (grown in dextrose) states. As a control we labeled and tracked a single allele with both colors of fluorescent proteins integrated ∼12-18 kb apart. The dual-allele cases showed significant subpopulations of positive correlations that were larger than the 90th percentile value of the single-allele case (0.11), comprising 51% (26/51) in dextrose and 40% (34/86) in galactose. The presence of positive correlations indicates that gene copies are often closely associated in space and time, which may be consistent, for instance, with intertwined polymers or a mutual association with external protein machinery. Interestingly, we find both correlated and uncorrelated examples which appear peripherally confined, as the GAL genes are known to preferentially associate with the nuclear periphery upon activation.

Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast

The conserved mitotic kinase Bub1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, Bub1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both Bub1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of bub1Δ haploid cells and restores viability to bub1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both bub1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid bub1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither Bub1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B-independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that Bub1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B-mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments.

Cloning of the Zygosaccharomyces bailii GAS 1 homologue and effect of cell wall engineering on protein secretory phenotype

BackgroundZygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a β-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion.ResultsThe GAS1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS1 null mutant was able to restore the parental phenotype. The respective Z. bailii Δgas1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS1 gene with deletion cassettes having flanking regions of ~400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii Δgas1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1β secretion disruption had no relevant effect.ConclusionsThe data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as already widely reported in literature, our data confirmed that an "always working" solution to the problems related to recombinant protein production can be hardly, if never, found; instead, manipulations have to be finely tuned for each specific product and/or combination of host cell and product.

Genome-wide genetic analysis of polyploidy in yeast

Polyploidy, increased sets of chromosomes, occurs during development, cellular stress, disease and evolution. Despite its prevalence, little is known about the physiological alterations that accompany polyploidy. We previously described 'ploidy-specific lethality', where a gene deletion that is not lethal in haploid or diploid budding yeast causes lethality in triploids or tetraploids. Here we report a genome-wide screen to identify ploidy-specific lethal functions. Only 39 out of 3,740 mutations screened exhibited ploidy-specific lethality. Almost all of these mutations affect genomic stability by impairing homologous recombination, sister chromatid cohesion, or mitotic spindle function. We uncovered defects in wild-type tetraploids predicted by the screen, and identified mechanisms by which tetraploidization affects genomic stability. We show that tetraploids have a high incidence of syntelic/monopolar kinetochore attachments to the spindle pole. We suggest that this defect can be explained by mismatches in the ability to scale the size of the spindle pole body, spindle and kinetochores. Thus, geometric constraints may have profound effects on genome stability; the phenomenon described here may be relevant in a variety of biological contexts, including disease states such as cancer.

Chromosome Pairing Does Not Contribute to Nuclear Architecture in Vegetative Yeast Cells

There are several reports of a closer-than-random colocalization of homologous chromosomes in the vegetative nuclei of diploid budding yeast. Here, we studied by fluorescence in situ hybridization (FISH) the nuclear distribution of chromosomes and found a slight tendency toward closer proximity between homologous (allelic) loci than between any nonhomologous chromosomal regions. We show that most of this preferential association is not due to vegetative (also known as somatic) pairing but is caused by the polar orientation of interphase chromosomes (Rabl orientation). We quantified the occasional loss of detectable fluorescence signals that is inherent to the FISH method. Signal loss leads to the occurrence of a single signal that may be misinterpreted as the close association of two homologous chromosomal sites. The nuclear distribution of homologous loci, when corrected for the influence of nuclear architecture and methodological faults, was not different or was only marginally different from a random relative positioning as predicted by computer simulation. We discuss here several possibilities for the residual homologous proximity that do not invoke homology-dependent vegetative pairing, and we conclude that, in diploid budding yeast, constitutive vegetative pairing is a negligible factor for the organization of the interphase nucleus.

Transcriptional induction of repair genes during slowing of replication in irradiated Saccharomyces cerevisiae.

We investigated the inhibition of cell-cycle progression and replication and the induction of the transcriptional response in diploid budding yeast populations exposed to two different doses of gamma-rays resulting in 15 and 85% survival respectively. We studied the kinetics of the cellular response to ionizing treatment during the period required for all of the surviving cells to achieve at least one cell division. The length of these periods increased with the dose. Irradiated populations arrested as large-budded cells containing partially replicated chromosomes. The extent of the S-phase was proportional to the amount of damage and lasted 3 or 7h depending on the irradiation dose. In parallel to the division study, we carried out a kinetic analysis of the expression of 126 selected genes by use of dedicated microarrays. About 26 genes were induced by irradiation and displayed various pattern of expression. Interestingly, 10 repair genes (RAD51, RAD54, CDC8, MSH2, RFA2, RFA3, UBC5, SRS2, SPO12 and TOP1), involved in recombination and DNA synthesis, display similar regulation of expression in the two irradiated populations. Their pattern of expression were confirmed by Northern analysis. At the two doses, the expression of this group of genes closely followed the extended replication period, and their expression resumed when replication restarted. These results suggest that the damage-induced response and DNA synthesis are closely regulated during repair. The analysis of the promoter regions indicates a high occurrence of the three MCB, HAP and UASH regulatory boxes in the promoters of this group of genes. The association of the three boxes could confer an irradiation-replication specific regulation.

The unfolded protein response represses nitrogen-starvation induced developmental differentiation in yeast.

Diploid budding yeast exhibits two developmental programs in response to nitrogen starvation, pseudohyphal growth, and sporulation. Here we show that both programs are repressed by activation of the unfolded protein response (UPR), a stress-signal transduction pathway responsible for induction of endoplasmic reticulum (ER)-resident chaperones when protein folding in the ER is impaired. Pseudohyphal growth was derepressed in ire1Delta/ire1Delta and hac1Delta/hac1Delta strains. Activation of the UPR or overexpression of the transcription factor Hac1(i)p, the product of an unconventional splicing reaction regulated by the UPR, was sufficient for repression of pseudohyphal growth and meiosis. HAC1 splicing occurred in a nitrogen-rich environment but ceased rapidly on nitrogen starvation. Further, addition of ammonium salts to nitrogen-starved cells was sufficient to rapidly reactivate HAC1 splicing. We propose that high translation rates in a nitrogen-rich environment are coupled to limited protein unfolding in the ER, thereby activating the UPR. An activated UPR then represses pseudohyphal growth and meiosis. Nitrogen starvation slows translation rates, allowing for more efficient folding of nascent polypeptide chains, down-regulation of the UPR, and subsequent derepression of pseudohyphal growth and meiosis. These findings significantly broaden the range of physiological functions of the UPR and define a role for the UPR in nitrogen sensing.

Regulation of G2/M progression by the STE mitogen-activated protein kinase pathway in budding yeast filamentous growth.

Inoculation of diploid budding yeast onto nitrogen-poor agar media stimulates a MAPK pathway to promote filamentous growth. Characteristics of filamentous cells include a specific pattern of gene expression, elongated cell shape, polar budding pattern, persistent attachment to the mother cell, and a distinct cell cycle characterized by cell size control at G2/M. Although a requirement for MAPK signaling in filamentous gene expression is well established, the role of this pathway in the regulation of morphogenesis and the cell cycle remains obscure. We find that ectopic activation of the MAPK signal pathway induces a cell cycle shift to G2/M coordinately with other changes characteristic of filamentous growth. These effects are abrogated by overexpression of the yeast mitotic cyclins Clb1 and Clb2. In turn, yeast deficient for Clb2 or carrying cdc28-1N, an allele of CDK defective for mitotic functions, display enhanced filamentous differentiation and supersensitivity to the MAPK signal. Importantly, activation of Swe1-mediated inhibitory phosphorylation of Thr-18 and/or Tyr-19 of Cdc28 is not required for the MAPK pathway to affect the G2/M delay. Mutants expressing a nonphosphorylatable mutant Cdc28 or deficient for Swe1 exhibit low-nitrogen-dependent filamentous growth and are further induced by an ectopic MAPK signal. We infer that the MAPK pathway promotes filamentous growth by a novel mechanism that inhibits mitotic cyclin/CDK complexes and thereby modulates cell shape, budding pattern, and cell-cell connections.