Yeast Sporulation Research Articles

Histone Tail Cleavage as a Mechanism for Epigenetic Regulation.

Histones are essential for DNA packaging and undergo post-translational modifications that significantly influence gene regulation. Among these modifications, histone tail cleavage has recently garnered attention despite being less explored. Cleavage by various proteases impacts processes such as stem cell differentiation, aging, infection, and inflammation, though the mechanisms remain unclear. This review delves into recent insights on histone proteolytic cleavage and its epigenetic significance, highlighting how chromatin, which serves as a dynamic scaffold, responds to signals through histone modification, replacement, and ATP-dependent remodeling. Specifically, histone tail cleavage is linked to critical cellular processes such as granulocyte differentiation, viral infection, aging, yeast sporulation, and cancer development. Although the exact mechanisms connecting histone cleavage to gene expression are still emerging, it is clear that this process represents a novel epigenetic transcriptional mechanism intertwined with chromatin dynamics. This review explores known histone tail cleavage events, the proteolytic enzymes involved, their impact on gene expression, and future research directions in this evolving field.

APCCdh1-mediated degradation of Cdh1 is necessary for faithful meiotic chromosome segregation in S. cerevisiae.

The Anaphase-Promoting Complex/Cyclosome (APC/C) is a ubiquitin ligase that promotes the ubiquitination and subsequent degradation of numerous cell cycle regulators during mitosis and in G1. Proteins are recruited to the APC/C by activator proteins such as Cdh1. During the cell cycle, Cdh1 is subject to precise regulation so that substrates are not degraded prematurely. We have explored the regulation of Cdh1 during the developmental transition into meiosis and sporulation in the budding yeast S. cerevisiae. Transition to sporulation medium triggers the degradation of Cdh1. Cdh1 degradation is mediated by the APC/C itself in a "trans" mechanism in which one molecule of Cdh1 recruits a second molecule of Cdh1 to the APC/C for ubiquitination. Degradation requires an intact glucose-sensing SNF1 protein kinase complex (orthologous to the mammalian AMPK nutritional sensor), which directly phosphorylates Cdh1 on Ser-200 within an unstructured N-terminal region. In the absence of phosphorylation, expression of a Cdh1-S200A mutant is fully stabilized, leading to chromosome instability and loss of viability. We hypothesize that Cdh1 degradation is necessary for the preservation of cell cycle regulators and chromosome cohesion proteins between the reductional and equational meiotic divisions, which occur without the intervening Gap or S phases found in mitotic cell cycles.

Abstract 1437 Phosphatidate phosphatase Pah1 is required for sporulation in yeast

Abstract 1437 Phosphatidate phosphatase Pah1 is required for sporulation in yeast

Isoform switching regulates Swi/Snf activity in response to nutrients

Eukaryotic organisms have evolved complex gene regulatory networks to launch coordinated responses to external conditions and stimuli. Under environmental stress, such as nutrient depletion, these responses involve reallocation of cellular resources away from growth and cell cycle stimulating genes and towards stress‐responsive genes. This occurs through several mechanisms including changes at the transcriptional level, repression of ribosome biogenesis factors, altered translation initiation, and RNA features which modulate translation efficiency and transcript stability. Upon nutrient starvation in Saccharomyces cerevisiae, diploid cells undergo meiosis (sporulation in yeast). We have previously shown that the catalytic component of the Swi/Snf chromatin remodeling complex, Snf2, is responsible for shifting gene expression away from intron‐rich ribosomal protein genes (RPGs) to enhance splicing of meiotic intron‐containing genes (ICGs) during sporulation. A similar process occurs during the transition from growth to quiescence known as the diauxic shift in yeast. During both of these complex cell‐state transitions, Snf2 levels change dramatically while SNF2 mRNA levels remain relatively stable. Although critical to its activity, the mechanism by which Snf2 protein levels change remains unknown. Here we describe temporally‐regulated alternate transcription start sites (TSS) under batch growth conditions, which produce SNF2 transcripts with different 5’ leaders affecting downstream translation propensity. Specifically, a long isoform of SNF2 contains three upstream open reading frames (uORFs), which we hypothesize inhibits the translation of the downstream protein‐coding ORF. I used 5’ RACE to identify the previously unannotated TSS of the long isoform, and RNA analysis suggests that the relative levels of each transcript are dynamic and affected by the binding of the highly‐conserved transcriptional regulators Ume6 and Cbf1 at the SNF2 locus. Protein analysis via Western blotting shows that this regulation indeed affects Snf2 expression. In light of the conservation of Snf2, investigating its regulation in response to environmental changes in S. cerevisiae may carry important implications for Snf2 chromatin remodeling activity in higher eukaryotes.

The Smk1 MAPK and Its Activator, Ssp2, Are Required for Late Prospore Membrane Development in Sporulating Saccharomyces cerevisiae.

During sporulation in the budding yeast Saccharomyces cerevisiae, proper development of the prospore membrane is necessary for the formation of viable spores. The prospore membrane will eventually become the plasma membrane of the newly formed haploid spore and also serves as the template for the deposition of the spore wall. The prospore membrane is generated de novo during meiosis II and the growing edge of the prospore membrane is associated with the Leading Edge Protein (LEP) complex. We find that the Smk1 MAP kinase, along with its activator Ssp2, transiently localizes with the LEP during late meiosis II. SSP2 is required for the leading edge localization of Smk1; this localization is independent of the activation state of Smk1. Like other LEP components, the localization of Smk1 at the leading edge also depends on Ady3. Although prospore membrane development begins normally in smk1 and ssp2 mutants, late prospore membrane formation is disrupted, with the formation of ectopic membrane compartments. Thus, MAP kinase signaling plays an important role in the formation of the prospore membrane.

Systematic genetic and proteomic screens during gametogenesis identify H2BK34 methylation as an evolutionary conserved meiotic mark

BackgroundGametes are highly differentiated cells specialized to carry and protect the parental genetic information. During male germ cell maturation, histone proteins undergo distinct changes that result in a highly compacted chromatin organization. Technical difficulties exclude comprehensive analysis of precise histone mutations during mammalian spermatogenesis. The model organism Saccharomyces cerevisiae possesses a differentiation pathway termed sporulation which exhibits striking similarities to mammalian spermatogenesis. This study took advantage of this yeast pathway to first perform systematic mutational and proteomics screens on histones, revealing amino acid residues which are essential for the formation of spores.MethodsA systematic mutational screen has been performed on the histones H2A and H2B, generating ~ 250 mutants using two genetic backgrounds and assessing their ability to form spores. In addition, histones were purified at key stages of sporulation and post-translational modifications analyzed by mass spectrometry.ResultsThe mutation of 75 H2A H2B residues affected sporulation, many of which were localized to the nucleosome lateral surface. The use of different genetic backgrounds confirmed the importance of many of the residues, as 48% of yeast histone mutants exhibited impaired formation of spores in both genetic backgrounds. Extensive proteomic analysis identified 67 unique post-translational modifications during sporulation, 27 of which were previously unreported in yeast. Furthermore, 33 modifications are located on residues that were found to be essential for efficient sporulation in our genetic mutation screens. The quantitative analysis of these modifications revealed a massive deacetylation of all core histones during the pre-meiotic phase and a close interplay between H4 acetylation and methylation during yeast sporulation. Methylation of H2BK37 was also identified as a new histone marker of meiosis and the mouse paralog, H2BK34, was also enriched for methylation during meiosis in the testes, establishing conservation during mammalian spermatogenesis.ConclusionOur results demonstrate that a combination of genetic and proteomic approaches applied to yeast sporulation can reveal new aspects of chromatin signaling pathways during mammalian spermatogenesis.

Investigating the Effects of Gpa2 Phosphorylation on Yeast Sporulation

Gpa2 is a G alpha protein in budding yeast, Saccharomyces cerevisiae, that functions in the nutrient signaling pathway. In diploid yeast cells, Gpa2 interacts with the cytoplasmic protein Ime2 and acts as an inhibitor to prevent Ime2 from inappropriate activation of sporulation. It is unclear how the inhibitory effect of Gpa2 on Ime2 is regulated. Recently, we found that Gpa2 undergoes phosphorylation and that its level of phosphorylation increases upon nitrogen starvation, a condition that triggers sporulation in yeast. Interestingly, phosphorylation enhances plasma membrane localization of Gpa2, raising the possibility that Gpa2 phosphorylation may play a role in the regulation of yeast sporulation. Accordingly, we investigated the sporulation efficiency of diploid cells that express either wild type Gpa2 or Gpa2‐10SA mutant, which lacks 10 serine residues required for phosphorylation. Using confocal microscopy to analyze the cells with clear indicators of the presence of spores, we found that more cells expressing wild type Gpa2 underwent sporulation than cells expressing Gpa2‐10SA mutant did. Our findings suggest that regulated phosphorylation of Gpa2 is a potential mechanism that relieves the inhibitory effect of Gpa2 on Ime2.Support or Funding InformationKnoedler Undergraduate Fund from Saint Louis University

Yeast Sporulation and [SMAUG+] Prion: Faster Is Not Always Better.

Chakravarty etal. (2019) and Itakura etal. (2019) report that the yeast RNA-binding protein Vts1 can convert into the [SMAUG+] prion state and delay meiosis commitment in response to starvation. It enables budding yeast to optimize their sporulation efficiency depending on how quickly nutrient availability fluctuates in their environment.

Protein dynamic analysis of the budding yeast sporulation process at the single-cell level in an air-enriched microfluidic device

Protein dynamic analysis of the budding yeast sporulation process at the single-cell level in an air-enriched microfluidic device

Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast.

Extant species have wildly different numbers of chromosomes, even among taxa with relatively similar genome sizes (for example, insects)1,2. This is likely to reflect accidents of genome history, such as telomere-telomere fusions and genome duplication events3-5. Humans have 23 pairs of chromosomes, whereas other apes have 24. One human chromosome is a fusion product of the ancestral state6. This raises the question: how well can species tolerate a change in chromosome numbers without substantial changes to genome content? Many tools are used in chromosome engineering in Saccharomyces cerevisiae7-10, but CRISPR-Cas9-mediated genome editing facilitates the most aggressive engineering strategies. Here we successfully fused yeast chromosomes using CRISPR-Cas9, generating a near-isogenic series of strains with progressively fewer chromosomes ranging from sixteen to two. A strain carrying only two chromosomes of about six megabases each exhibited modest transcriptomic changes and grew without major defects. When we crossed a sixteen-chromosome strain with strains with fewer chromosomes, we noted two trends. As the number of chromosomes dropped below sixteen, spore viability decreased markedly, reaching less than 10% for twelve chromosomes. As the number of chromosomes decreased further, yeast sporulation was arrested: a cross between a sixteen-chromosome strain and an eight-chromosome strain showed greatly reduced full tetrad formation and less than 1% sporulation, from which no viable spores could be recovered. However, homotypic crosses between pairs of strains with eight, four or two chromosomes produced excellent sporulation and spore viability. These results indicate that eight chromosome-chromosome fusion events suffice to isolate strains reproductively. Overall, budding yeast tolerates a reduction in chromosome number unexpectedly well, providing a striking example of the robustness of genomes to change.

Histone tail cleavage as a novel epigenetic regulatory mechanism for gene expression.

Chromatin is an intelligent building block that can express either external or internal needs through structural changes. To date, three methods to change chromatin structure and regulate gene expression have been well-documented: histone modification, histone exchange, and ATP-dependent chromatin remodeling. Recently, a growing body of literature has suggested that histone tail cleavage is related to various cellular processes including stem cell differentiation, osteoclast differentiation, granulocyte differentiation, mammary gland differentiation, viral infection, aging, and yeast sporulation. Although the underlying mechanisms suggesting how histone cleavage affects gene expression in view of chromatin structure are only beginning to be understood, it is clear that this process is a novel transcriptional epigenetic mechanism involving chromatin dynamics. In this review, we describe the functional properties of the known histone tail cleavage with its proteolytic enzymes, discuss how histone cleavage impacts gene expression, and present future directions for this area of study.

A Role for the Respiratory Chain in Regulating Meiosis Initiation in Saccharomyces cerevisiae

Meiosis is a specific type of cell division that is essential for sexual reproduction in most eukaryotes. Mitochondria are crucial cellular organelles that play important roles in reproduction, though the detailed mechanism by which the mitochondrial respiratory chain functions during meiosis remains elusive. Here, we show that components of the respiratory chain (Complexes I-V) play essential roles in meiosis initiation during the sporulation of budding yeast, Saccharomyces cerevisiae Any functional defects in the Complex I component Ndi1p resulted in the abolishment of sporulation. Further studies revealed that respiratory deficiency resulted in the failure of premeiotic DNA replication due to insufficient IME1 expression. In addition, respiration promoted the expression of RIM101, whose product inhibits Smp1p, a negative transcriptional regulator of IME1, to promote meiosis initiation. In summary, our studies unveiled the close relationship between mitochondria and sporulation, and uncover a novel meiosis initiation pathway that is regulated by the respiratory chain.

Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials.

Effects of Temperature on the Meiotic Recombination Landscape of the Yeast Saccharomyces cerevisiae.

ABSTRACTAlthough meiosis in warm-blooded organisms takes place in a narrow temperature range, meiosis in many organisms occurs over a wide variety of temperatures. We analyzed the properties of meiosis in the yeast Saccharomyces cerevisiae in cells sporulated at 14°C, 30°C, or 37°C. Using comparative-genomic-hybridization microarrays, we examined the distribution of Spo11-generated meiosis-specific double-stranded DNA breaks throughout the genome. Although there were between 300 and 400 regions of the genome with high levels of recombination (hot spots) observed at each temperature, only about 20% of these hot spots were found to have occurred independently of the temperature. In S. cerevisiae, regions near the telomeres and centromeres tend to have low levels of meiotic recombination. This tendency was observed in cells sporulated at 14°C and 30°C, but not at 37°C. Thus, the temperature of sporulation in yeast affects some global property of chromosome structure relevant to meiotic recombination. Using single-nucleotide polymorphism (SNP)-specific whole-genome microarrays, we also examined crossovers and their associated gene conversion events as well as gene conversion events that were unassociated with crossovers in all four spores of tetrads obtained by sporulation of diploids at 14°C, 30°C, or 37°C. Although tetrads from cells sporulated at 30°C had slightly (20%) more crossovers than those derived from cells sporulated at the other two temperatures, spore viability was good at all three temperatures. Thus, despite temperature-induced variation in the genetic maps, yeast cells produce viable haploid products at a wide variety of sporulation temperatures.

Developmentally regulated internal transcription initiation during meiosis in budding yeast.

Sporulation of budding yeast is a developmental process in which cells undergo meiosis to generate stress-resistant progeny. The dynamic nature of the budding yeast meiotic transcriptome has been well established by a number of genome-wide studies. Here we develop an analysis pipeline to systematically identify novel transcription start sites that reside internal to a gene. Application of this pipeline to data from a synchronized meiotic time course reveals over 40 genes that display specific internal initiations in mid-sporulation. Consistent with the time of induction, motif analysis on upstream sequences of these internal transcription start sites reveals a significant enrichment for the binding site of Ndt80, the transcriptional activator of middle sporulation genes. Further examination of one gene, MRK1, demonstrates the Ndt80 binding site is necessary for internal initiation and results in the expression of an N-terminally truncated protein isoform. When the MRK1 paralog RIM11 is downregulated, the MRK1 internal transcript promotes efficient sporulation, indicating functional significance of the internal initiation. Our findings suggest internal transcriptional initiation to be a dynamic, regulated process with potential functional impacts on development.

Genome-scale fluxes predicted under the guidance of enzyme abundance using a novel hyper-cube shrink algorithm.

One of the long-expected goals of genome-scale metabolic modelling is to evaluate the influence of the perturbed enzymes on flux distribution. Both ordinary differential equation (ODE) models and constraint-based models, like Flux balance analysis (FBA), lack the capacity to perform metabolic control analysis (MCA) for large-scale networks. In this study, we developed a hyper-cube shrink algorithm (HCSA) to incorporate the enzymatic properties into the FBA model by introducing a pseudo reaction V constrained by enzymatic parameters. Our algorithm uses the enzymatic information quantitatively rather than qualitatively. We first demonstrate the concept by applying HCSA to a simple three-node network, whereby we obtained a good correlation between flux and enzyme abundance. We then validate its prediction by comparison with ODE and with a synthetic network producing voilacein and analogues in Saccharomyces cerevisiae. We show that HCSA can mimic the state-state results of ODE. Finally, we show its capability of predicting the flux distribution in genome-scale networks by applying it to sporulation in yeast. We show the ability of HCSA to operate without biomass flux and perform MCA to determine rate-limiting reactions. Algorithm was implemented by Matlab and C ++. The code is available at https://github.com/kekegg/HCSA. xiezhengwei@hsc.pku.edu.cn or qi@pku.edu.cn. Supplementary data are available at Bioinformatics online.

Yeast Sporulation Medium

Yeast Sporulation Medium

Selection of G1 Phase Yeast Cells for Synchronous Meiosis and Sporulation.

Centrifugal elutriation is a procedure that allows the fractionation of cell populations based upon their size and shape. This allows cells in distinct cell cycle stages can be captured from an asynchronous population. The technique is particularly helpful when performing an experiment to monitor the progression of cells through the cell cycle or meiosis. Yeast sporulation like gametogenesis in other eukaryotes initiates from the G1 phase of the cell cycle. Conveniently, S. cerevisiae arrest in G1 phase when starved for nutrients and so withdrawal of nitrogen and glucose allows cells to abandon vegetative growth in G1 phase before initiating the sporulation program. This simple starvation protocol yields a partial synchronization that has been used extensively in studies of progression through meiosis and sporulation. By using centrifugal elutriation it is possible to isolate a homogeneous population of G1 phase cells and induce them to sporulate synchronously, which is beneficial for investigating progression through meiosis and sporulation. An additionally benefit of this protocol is that cell populations can be isolated based upon size and both large and small cell populations can be tested for progression through meiosis and sporulation. Here we present a protocol for purification of G1 phase diploid cells for examining synchronous progression through meiosis and sporulation.

Lipid droplets are central organelles for meiosis II progression during yeast sporulation.

Neutral lipids, predominantly triacylglycerol (TAG) and sterol ester, are stored within the cellular organelles termed lipid droplets (LDs). Although it is believed that the major function of LDs is to supply the cell with energy and membranes, little is known about the cellular events directly involving LDs and their contents. In this study, we provide cytological evidence that LDs form direct contacts with the prospore membrane (PSM) that is synthesized de novo during meiosis II to sequester the dividing nuclei in sporulating yeast. Lipidomic analyses indicate that TAG lipolysis releases free fatty acids at a time that correlates well with meiosis II progression, concomitant with phospholipid remodeling. Mutants lacking TAG or impaired of TAG hydrolysis show spore wall assembly defects, supporting a role for TAG and/or its metabolites in spore wall morphogenesis. Not only does LD integrity influence spore wall assembly, LDs are also essential for other aspects of spore development. Yeast cells lacking LDs are severely defective in PSM growth and organization and display disrupted spindles, producing dead spores or even failing to form spores. Together these results link LD physiology directly to a unique membrane morphogenesis process critical for development.

Different Toxicity Mechanisms for Citrinin and Ochratoxin A Revealed by Transcriptomic Analysis in Yeast.

Citrinin (CIT) and ochratoxin A (OTA) are important mycotoxins, which frequently co-contaminate foodstuff. In order to assess the toxicologic threat posed by the two mycotoxins separately or in combination, their biological effects were studied here using genomic transcription profiling and specific live cell gene expression reporters in yeast cells. Both CIT and OTA cause highly transient transcriptional activation of different stress genes, which is greatly enhanced by the disruption of the multidrug exporter Pdr5. Therefore, we performed genome-wide transcription profiling experiments with the pdr5 mutant in response to acute CIT, OTA, or combined CIT/OTA exposure. We found that CIT and OTA activate divergent and largely nonoverlapping gene sets in yeast. CIT mainly caused the rapid induction of antioxidant and drug extrusion-related gene functions, while OTA mainly deregulated developmental genes related with yeast sporulation and sexual reproduction, having only a minor effect on the antioxidant response. The simultaneous exposure to CIT and OTA gave rise to a genomic response, which combined the specific features of the separated mycotoxin treatments. The application of stress-specific mutants and reporter gene fusions further confirmed that both mycotoxins have divergent biological effects in cells. Our results indicate that CIT exposure causes a strong oxidative stress, which triggers a massive transcriptional antioxidant and drug extrusion response, while OTA mainly deregulates developmental genes and only marginally induces the antioxidant defense.