Fission Yeast Schizosaccharomyces pombe: A Unicellular “Micromammal” Model Organism

Abstract

The fission yeast Schizosaccharomyces pombe is a rod-shaped unicellular eukaryote, well known for its contributions as a model organism to our understanding of regulation and conservation of the eukaryotic cell cycle. As a yeast divergent from the budding yeast Saccharomyces cerevisiae, S. pombe shares more common features with humans including gene structures, chromatin dynamics, and the prevalence of introns, as well as the control of gene expression through pre-mRNA splicing, epigenetic gene silencing, and RNAi pathways. With the advent of new methodologies for research, S. pombe has become an increasingly used model to investigate various molecular and cellular processes over the last 50 years. Also, S. pombe serves as an excellent system for undergraduate students to obtain hands-on research experience. Versatile experimental approaches are amenable using the fission yeast system due to its relative ease to maintain, its inherent cellular properties, its power in classic and molecular genetics, and its feasibility in genomics and proteomics analyses. This article provides an overview of S. pombe’s rise as a valuable model organism and presents examples to highlight the significance of S. pombe as a unicellular “micromammal” in investigating biological questions. We especially focus on the advantages of and the advancements in using fission yeast for studying biological processes that are characteristic of metazoans, to decipher the underlining molecular mechanisms fundamental to all eukaryotes.

INTRODUCTION

A Swahili beer.

Although the fission yeast Schizosaccharomyces pombe is not known for its use in baking as the budding yeast Saccharomyces cerevisiae is, it has been used in brewing. Pombe is the word for “beer” in Swahili, representing a fermented beverage that originated in East Africa. S. pombe was initially isolated from contaminated millet beer by Saare and colleagues. It is wildly present in various natural sources such as fruits, syrups, and a type of tea, “Kombucha,” produced by fermentation with yeasts and bacteria. It is also used for making distilled spirits, as well as for reducing acidity in wine. S. pombe was subsequently purified in the early 1890s by Paul Lindner and colleagues. To distinguish it from Saccharomyces cerevisiae, Lindner named the organism Schizosaccharomyces pombe. Schizosaccharomyces indicates that it is an ascomycete yeast belonging to the Fungi kingdom, as well as it reproduces via fission and lives as a unicellular eukaryote with the ability to ferment sugars (Fantes & Hoffman, 2016; Gomes et al., 2002; Hayles & Nurse, 2016; Hoffman, Wood, & Fantes, 2015). Paul Nurse, a pioneer in the field of cell cycle using S. pombe as a model organism, visited a jungle brewery in Uganda to taste pombe and wrote: “The taste was sweet and very alcoholic but definitely drinkable” (Hayles & Nurse, 2016).

A research model.

S. pombe may not make a popular beer, but it has proven to be an excellent experimental model organism for investigating biological questions. Unlike the investigations in S. cerevisiae, which began in order to improve brewing and baking before the modern age, research in S. pombe has been driven by curiosity and biological questions from the beginning. Swiss scientist Urs Leupold investigated the genetic basis of the mating-type system in S. pombe for his Ph.D. thesis in the 1940s. He isolated the ancestor strains, 968 h⁹⁰, 972 h⁻, and 975 h⁻, from which almost all currently used modern strains have been derived. Leupold not only founded S. pombe as a genetically tractable model organism but also established the basis for the nearly isogenic background of all the strains used in research, thereby enhancing the consistency of data collected from different labs worldwide. During the same period, Murdoch Mitchison was interested in understanding the relationship between cell growth and cell division. After exploring different organisms, he chose S. pombe, as it grows by extension and divides symmetrically, providing a convenient system to monitor the cell length/size with respect to the cell cycle stages. In the 1970s, Paul Nurse employed genetic methods that he had learned in Leupold’s lab at the University of Bern for isolating fission yeast cell division cycle (cdc) mutants in Mitchison’s laboratory at Edinburgh University — the integration between genetics and cell biology in S. pombe research (Fantes & Hoffman, 2016; Hoffman et al., 2015). This model organism has now been used worldwide in different biological fields, thereby forming an international S. pombe research community.

A different yeast.

In addition to their distinct biological characteristics (Figure 1), accumulated evidence support fission yeast S. pombe as a “micromammal,” more similar to complex eukaryotes than budding yeast S. cerevisiae. First, although they are both ascomycete yeasts and unicellular eukaryotes, S. pombe and S. cerevisiae evolutionarily diverged from each other approximately 350-400 million years ago (Sipiczki, 2000; Wood et al., 2002) (Figure 2). Since the evolutionary distance between these two yeasts is in the same order as that between yeast and mammals, S. pombe and S. cerevisiae are not closer to each other, than they are to humans. Second, according to the protein and DNA sequence data, S. pombe represents a more “basal” group than S. cerevisiae and retains more common ancestral features after the split between fungi and metazoans (Hoffman et al., 2015). In contrast, S. cerevisiae has evolved more rapidly than S. pombe and lost more than 300 genes and several biological processes that are conserved between S. pombe and complex eukaryotes (Aravind, Watanabe, Lipman, & Koonin, 2000; Wood, 2006). Furthermore, chromosomal sequence analysis has not revealed evidence of large-scale genome duplications in S. pombe as seen in other eukaryotes such as S. cerevisiae and Arabidopsis (Wood et al., 2002). Out of 5,064 total protein-coding genes in the fission yeast, 3,536 of them have human homologs and 1,244 are disease-associated genes (pombase.org), making S. pombe a valuable system to assess the potential function of genes from complex eukaryotes such as humans in the context of an intact cell under physiological conditions (Rine, 2014).

Figure 1. Comparison of fission yeast S. pombe and budding yeast S. cerevisiae morphology.

Left panel, S. pombe cells in fluorescence microscopy with visible nuclei (red, DAPI staining), septa (blue, Calcofluor staining), and a GFP-tagged nuclear (image obtained by Tang lab). Right panel, S. cerevisiae cells in DIC microscopy (source: https://en.wikipedia.org/wiki/Yeast#/media/File:S_cerevisiae_under_DIC_microscopy.jpg).

Figure 2. Consensus phylogenetic tree tracing the divergent distance between S. pombe and S. cerevisiae.

S. cerevisiae has undergone more divergent events and is phylogenetically more distant than S. pombe to their common ancestor at the metazoan-fungi branching point. Modified and adapted from (Lertwattanasakul et al., 2015; Sipiczki, 2000).

For all the facts stated above, combined with the development of new investigative methodologies, S. pombe has increasingly been used as a model organism over the last century. The large evolutionary distance between S. pombe and S. cerevisiae suggests that if a gene or biological process is conserved in both yeasts, it is likely to have homologous genes in mammals including humans. Thus, adding S. pombe as another yeast to experimental systems enables us to make comparisons between the two divergent yeast in an evolutionary context and decipher the conservation of a gene function or a biological process of interest in all eukaryotes including humans. On the other hand, variations between the two yeasts in cellular processes, such as the different steps of gene expression, allows us to determine whether and how alternative mechanisms are evolved to mitigate environmental challenges.

As eukaryotic models, S. pombe and S. cerevisiae share their suitability for classical and molecular genetics, as well as for systems biology approaches. The fission yeast S. pombe has become an attractive research model as it retains several conserved cellular processes, such as cell-cycle organization, gene and chromosome structures involving telomeres, centromeres, origin of replication, and the prevalence of introns (Hayles & Nurse, 2016; Kim et al., 2010). In addition to its particular impact on the studies of cell-cycle regulation and DNA damage checkpoint and repair mechanisms, S. pombe as a eukaryotic model organism offers advantages for investigating the biological processes that have been lost in S. cerevisiae through evolution, including chromosome dynamics related to epigenetic gene silencing (White & Allshire, 2008) and gene expression control via pre-mRNA processing (Hayles & Nurse, 2016; Wood et al., 2002). We present examples in this paper to further highlight the significance of S. pombe as a unicellular “micromammal” model organism in biological research.

BIOLOGY OF S. POMBE

Life Cycle

The fission yeast S. pombe is a rod-shaped unicellular eukaryote. It can be grown at 25-36 °C in minimal and/or rich media with a generation time ranging from 2-4 hours. Table 1 provides general information about the morphology, genome, and growth conditions of S. pombe cells. Although most yeast strains used in the laboratory are haploid strains, both the fission yeast S. pombe and the budding yeast S. cerevisiae can reproduce either in a diploid or a haploid state. However, in a natural environment, budding yeast tends to live as a diploid, whereas fission yeast tends to be in the haploid form (Forsburg, 2003). Figure 3 depicts the life cycle of S. pombe through the vegetative (mitotic) as well as sexual (meiotic) reproductive stages in both haploid (3A) and diploid states (3B).

Table 1.

Common morphological and genetic characteristics of S. pombe

Common Morphology
Shape	Rod-like
Length*	7-14 microns
Width*	3-4 microns
Spore Type	Ascus (sac-like) spore
Genetic Characteristics
Genome Size	12.6 million bp
No. of Chromosomes	Three Chromosomes: Chromosome I (5.6 Mb) Chromosome II (4.5 Mb) Chromosome III (2.4 Mb)
Identified Protein Encoding Genes	5,118 (3,539 Homologous to H. sapiens)
Growth Conditions
Average Doubling Time	2 – 4 hours in culture
Culture Temperature	25 °C – 36 °C
Cell spectrometry	O.D of 1 @595nm = 1 × 107 cells

^*These dimensions are reflective of haploid cells. Diploids are wider and longer than haploids.

Figure 3. Summary of the life cycle of S. pombe in both haploid and diploid states, through the vegetative as well as sexual reproduction stages.

A. Haploid life cycle of the fission yeast S. pombe. Fission yeast cells are usually in the haploid life cycle and can shuttle between the vegetative and sexual reproductive (meiotic) cycles. Conjugation can be triggered between cells of opposite mating types (h⁺ and h⁻) through stress such as nutrient deprivation, resulting in the brief generation of a zygote that can undergo the meiotic cycle to produce four zygotic spores once nutrient availability improves. However, if the conditions are controlled such that the conjugated zygote is provided with nutrients, it can enter and sustain itself as a diplontic cell. B. Diploid life cycle of the fission yeast, S. pombe. The diplontic S. pombe cell can be sustained and propagated under laboratory conditions. The diploid fission yeast can also shuttle between the vegetative and meiotic cycles. Once it enters the meiotic cycle, however, it results in haploid spores that can then re-enter the haplontic life cycle.

Under conditions of nutrient deprivation, S. pombe can be triggered to enter the meiotic cycle (Egel, 1971). For conjugation, it is essential to have two haploid cells of different mating types, h⁺ and h⁻, in close physical proximity to one another. Once conjugated, the cells enter a transient diploid zygotic stage (Figure 3A). The zygote then initiates meiosis, leading to four haploid nuclei. Each of these nuclei develops a spore wall called an ascus within the zygote. The tetrad ascus has a distinct bent shape (zygotic ascus). When the environment is preferable for cell growth, the zygote wall lyses and the homologous chromosomes undergoes potential recombination events. As a result, four new haploid cells are produced to proceed into vegetative growth through the mitotic cell cycle (Hayles & Nurse, 2016; Tang, 2016). If the two haploid cells are supplied with nutrients soon after their conjugation, the resulting diploid cell can be recovered and maintained in the vegetative diploid cycle, especially in laboratory conditions. A diploid cell is meiosis competent only if it inherits a mating-type composition of h⁺/h⁻, and thus may be triggered to undergo meiosis. This diploid cell follows a similar trajectory into post-zygotic meiosis (Figure 3B) to generate an ascus containing four haploid ascospores (Egel & Egel-Mitani, 1974). However, the morphology of the ascus produced from a diploid, azygotic ascus, is linear; this morphology is distinctly different from the haploid, zygotic ascus, which is bent as it enters the meiotic cycle (Figure 3, compare the zygotic and azygotic ascus in panels A and B).

Mitotic Cell Cycle

The mitotic cell cycle of the fission yeast S. pombe consists of consecutive G₁, S, G₂, and M phases, typical to that of metazoans (Figure 4A). For the fission yeast, G₂ is normally the longest phase (Forsburg & Rhind, 2006). In contrast, budding yeast S. cerevisiae cells spend most of their time in G₁ phase, with a less distinctive G₂ (Figure 4B, (Alberts, 2007). The different features of the cell cycle between the two yeasts determine their variant control points in the cell cycle: the G₂/M transition acts as a major visible control in the S. pombe cell cycle, while the G₁/S transition—known as Start, serves as a key restriction in the S. cerevisiae cell cycle. This explains why most of the cell-division cycle (cdc) mutants originally isolated from fission yeast and budding yeast arrest in G₂ and G₁, respectively (Figure 4A and 4B) at the restrictive temperature (Hartwell, Culotti, Pringle, & Reid, 1974; Hartwell, Mortimer, Culotti, & Culotti, 1973; Nurse, Thuriaux, & Nasmyth, 1976). S. pombe thus has been historically used as a model organism for genetically dissecting the control circuit at G₂/M transition of the cell cycle.

Figure 4. A comparison of the mitotic cell cycles between S. pombe and S. cerevisiae.

A. The fission yeast follows the conventional mitotic cycle with distinct, consecutive G₁, S, G₂, and M phases. A cell typically spends the longest time in the G₂ phase (~70-80%). The daughter nuclei segregate and initiate G₁; however, cytokinesis and the final separation of the new cells do not take place until the S-phase. The G₂–M transition is usually the major control point for the cell cycle (arrow). Most cell-division cycle (cdc) mutants are blocked at G₂, and thus are incapable of entering mitosis, displaying a cell elongation phenotype at a restrictive temperature. cdc2^ts encodes a master protein kinase that is involved in cell cycle regulation. B. The budding yeast cell cycle features distinct G₁ and S phases; the G₂ and M phases are not clearly defined. The G₁–S transition is the major control point for the cell cycle (arrow). CDC28^ts encodes a Cdc2 master protein kinase homolog; at the restrictive temperature, this mutant cannot progress into S-phase, exhibiting a non-budding phenotype.

Interestingly, the G₁/S control can also be revealed in fission yeast mutants, such as wee1⁻ (Figure 5). Normally, the G₂/M transition provides the evident and major size control point. Since each cell division gives rise to two daughter cells of a larger size than that required to pass G₁/S, the control point — Start, is invisible in wild-type cells (Figure 4A). However, wee1 mutant cells enter mitosis prematurely and produce new cells smaller than that needed for passing through Start, which triggers the size control at G₁/S transition and unravels additional restriction points in the S. pombe cell cycle (Hayles & Nurse, 2016; Nurse & Thuriaux, 1980). Therefore, as in complex eukaryotes, G₁/S transition and G₂/M transition are two control points that regulate the S. pombe cell cycle to ensure that cells do not initiate DNA replication or enter mitosis, respectively, unless conditions are favorable. The cyclin-dependent serine/threonine protein kinase CDK1 (humans)/Cdc2 (S. pombe) is required for the timing and size control at which cells progress through both G₁/S and G₂/M transitions in S. pombe (Hayles & Nurse, 2016).

Figure 5. A wee mutant mitotic cell cycle.

Newly divided wt cells are of greater size than that required to enter S phase, and thus spend minimal time in G₁ (Figure 4A). In contrast, wee mutant cells enter mitosis at a size that is smaller than that required for undergoing G₁/S. They need to spend more time in G₁ to attain a standard size and then pass through G₁/S. The major control point of cell cycle progression is the G₁/S transition (arrow). Therefore, the total cell cycle length is the same for a wild type or a wee mutant strain, the difference is the alteration in the length of G₁ and G₂.

Notably, as yeasts, S. pombe and S. cerevisiae share the common process of closed mitosis, in which the nuclear envelope does not break down, as it usually does in most multi-cellular animal cells exhibiting a morphology known as open mitosis. In particular, since fission yeast cells do not complete cytokinesis until the end of G₁ in the following cycle, each cell contains a 2C DNA content through most of the cell cycle stages.

Importantly, a key life feature that renders S. pombe a powerful model for cell cycle study is its growth polarity and symmetrical dividing pattern. Unlike S. cerevisiae that divides by asymmetrical budding, S. pombe grows by tip elongation and divides by medial fission, while its cell diameter remains relatively unchanged. Growth polarity allows the cell cycle stages to be followed conveniently and precisely by measuring cell size, specifically cell length, thereby permitting detailed analysis of the control in cell cycle progression. The remarkable contributions of S. pombe as a model system for the discovery of fundamental mechanisms governing cell cycle regulation conserved through evolution in eukaryotes will be elaborated further in NOTABLE DISCOVERIES.

Mating Type Locus

The birth of S. pombe as a research model organism was during Leupold’s endeavor to identify the genetic mating type locus in this species, which we now know as a complex region harboring expressed and silenced genes. A major factor dictating conjugation is the presence of two different pombe mating types. A specific mating type in S. pombe is defined by the expression of either the P allele or the M allele from the mating-type locus (Leupold, 1950). The mating-type locus is on Chromosome 2 and consists of three transcriptional loci, mat1, mat2-P, and mat3-M (D. H. Beach & Klar, 1984). The mat2-P and mat3-M alleles act as information donors for mat1 locus (Figure 6). In homothallic strains, i.e. those that can find conjugation partners in the same culture, the mat1 locus can undergo spontaneous switching with either the mat2 or the mat3 locus via a replication-recombination event (D.H. Beach, 1983; Egel, Beach, & Klar, 1984). Thus, the strain expresses either the P or the M factor, as a result of the switching, respectively. The most commonly used homothallic strain is the h⁹⁰ strain, which may switch its mating type at the mat1 locus every 3 generations (>90% of cells in a culture at a time). Thus, about half the cells in the culture of h⁹⁰ mating type strain would express the P factor while the other half express the M factor, facilitating conjugation.

Figure 6. The arrangement and expression of the differing alleles from the mating-type locus in the fission yeast S. pombe.

There are three commonly used mating type strains for S. pombe—h⁹⁰, h⁺, and h⁻ —which are the result from the differential activity at three transcriptional loci on chromosome two: mat1, mat2, and mat3. In all strains, the alleles at mat2 and mat3 loci are silenced but can influence expression from the mat1 locus. In heterothallic strains, either mat2 (in h⁺ strains) or mat3 (in h⁻strains) affect the expression from the mat1 locus to express only the plus (P) or the minus (M) allele, respectively. In homothallic strains, the mat1 loci can express either the plus or minus allele due to a recombination event, known as switching, resulting in an equal mixture of cells expressing either the P or the M allele in the cell culture.

In contrast to the homothallic strains, heterothallic strains are restricted to only expressing either the P or M factor from the mat2 or mat3 locus, respectively (Egel et al., 1984). The two most commonly used heterothallic strains are 972 h⁻ and 975 h⁺. The h⁺ strain only has a transcriptionally active mat2P locus, while the h⁻ strain is only transcriptionally active at its mat3M locus for the informational switch with mat1. Figure 6 illustrates the arrangement and expression of the differing alleles from the mating-type locus in the fission yeast S. pombe.

Nutritional starvation condition induces the heterothallic mating types, h⁺ and h⁻ strains, to conjugate with each other and undergo meiotic sporulation. Similar conditions also trigger the homothallic mating-type strain, h⁹⁰, to either mate with itself or with either of the heterothallic strains. However, zygotic diploids are only formed between strains with opposite mating types h⁺ and h⁻ from heterothallic strains or homothallic h⁹⁰ strains. A diploid homozygous for either h⁺ or h⁻ is incompetent for meiosis and sporulation (Hoffman et al., 2015).

The ability of S. pombe to alternate between haploid and diploid states in response to different conditions, provides a powerful tool for research in this experimental system. Thus, the mating behavior of S. pombe can be manipulated in the laboratory to shift the organism’s ploidy state through simple changes of its reproduction environment. In a haploid strain that harbors a loss-of-function mutation of a gene, the recessive trait can be readily displayed under proper conditions, such as a restrictive temperature, which would be otherwise masked due to the presence of a dominant, wild-type allele in a diploid strain. This technique has proved to be a powerful strategy for genetically dissecting many cellular processes, most notably the components in the regulatory circuits of the cell cycle. On the other hand, using the haploinsufficiency assay in a diploid strain, a specific gene dosage effect can be assessed by determining the changes in the functional state between heterozygous and homozygous strains for the gene. The inherent biological features of S. pombe that offer the controlled regulation of dual ploidy states are further elaborated in the later sections (Forsburg, 2003; Kim et al., 2010).

Chromosomal Organization and Chromatin Modification

Both S. pombe and S. cerevisiae have a small genome of approximately 12 Mbp—about 1/250^th of the 3,100 Mbp human genome—and thus are considered the most efficient eukaryotes. However, despite possessing similarly sized genomes, the S. pombe chromatin has many features that are characteristic of mammalian cells, but that are missing or changed in budding yeast (Forsburg, 2003; Wood et al., 2002). Fission yeast displays higher order complexity in its telomeric organization, a feature that is comparable to their metazoic counterparts. The 300 bp stretch of fission yeast telomeres contain repeating units of 8-11 bp conserved sequences, and end with a single stranded 5’overhang sequence. The telomeric regions in Chromosome I and II are further flanked by 20-40 kb of repetitive subtelomeric (STE) sequences. Although no subtelomeric sequences have been identified in Chromosome III, approximately ~1.2 Mb of ribosomal DNA is harbored between both its arms. Similarly, the origin of replication (oriC) sites in fission yeast show conserved higher order features. While there are 1200 oriC sites in S. pombe, only a subset of these is used in every replication cycle. Like the oriC sites of many metazoans, those in S. pombe vary in both their length (0.5 to 3 kb) and in their replication efficiency. In addition to the retention of conserved metazoan chromosomal structural features such as telomeres and DNA replication origins, the centromeres of S. pombe are much larger (Cen I – 35 Kbp, Cen II – 65 Kbp, and Cen III – 110 Kbp) and more complex than the 125 bp centromere of S. cerevisiae (Figure 7). The centromeric region of all three chromosomes in fission yeast consists of a ~ 4kb central core (cc) region, flanked by two distinct repetitive domains on either side. The cc domain is associated with the centromere-specific histone H3 variant, CENP-A (Cam & Whitehall, 2016). Immediately flanking the cc sequences are the innermost repeats (imr) regions, while the outer repeat (otr) sequences set the boundaries of the centromeric heterochromatin. The typical centromeric arrangement of the otr region consists of a varying number of dg and dh repetitive elements (Niwa, Matsumoto, Chikashige, & Yanagida, 1989; Wood et al., 2002). As shown in Figure 7, other than the complexity, the arrangement of repetitive sequences in the centromeres and flanking regions in S. pombe is also similar to those in humans but is absent in S. cerevisiae (Allshire & Ekwall, 2015; Allshire & Karpen, 2008; Matsuda, Asakawa, Haraguchi, & Hiraoka, 2017; Wood et al., 2002).

Figure 7. Schematic representations of the centromeric organizations and histone arrangements in S. cerevisiae, S. pombe, and H. sapiens.

S. cerevisiae displays a very short centromeric region with no distinct heterochromatinization. The S. pombe centromere, on the other hand, has a central domain consisting of a central core flanked by innermost repeat (imr) regions. The outer repeat regions (otr) lie on either side of the central domain and are primarily associated with the heterochromatinization of the centromere. The centromeric arrangement in human chromosomes spans a larger region, containing multiple repeat units associated with methylated histone variants resulting in a large heterochromatin region at the centromere. Overall, the centromeric arrangement in fission yeast, with distinctive repeats and heterochromatinization, is much closer to the organization observed in humans. Colored circles above the centromeric sequences are representative of the enriched histone type associated with the centromeric domain. Centromere lengths, histone size, and histone numbers are not to scale. Broad arrows represent various repetitive elements at centromeres. Adapted based on (Allshire & Karpen, 2008).

Chromatin structure is an important component of the epigenetic regulation of gene expression. Fission yeast bears a high degree of conservation with metazoans in post-translational modifications (PTMs) of histone proteins involved in heterochromatin formation, maintenance, and spreading. For example, telomere-binding proteins such as Taz1 and Pot1 (Nandakumar & Cech, 2013), the H3K9 methylating protein Clr4, and the HP1 family of heterochromatin proteins including Swi6 (Grewal, 2010) are all functional homologs of human proteins in S. pombe.

A remarkable difference between the two yeasts is the existence of the RNAi pathways in S. pombe, as is in complex eukaryotes, but not in S. cerevisiae. Moreover, heterochromatin assembly at the centromeric repetitive sequences is directed by the RNAi machinery in fission yeast. The RNAi pathway in S. pombe is required for recruiting the centromere-specific H3 variant, CENP-A, to the central core and forming the kinetochore to accomplish chromosome segregation at mitosis, which is the primary function of the centromere (French & Straight, 2013).

The highly conserved components of the RNAi machinery— namely Dicer (Dcr1 RNase), Argonaute (Ago1 DNA binding protein), and the RNA-directed RNA polymerase Rbp1 have been identified and found necessary for establishing heterochromatin in S. pombe (Zofall & Grewal, 2006). Briefly, Dicer mediates the conversion of double-stranded RNA transcribed from repetitive regions into small interfering RNAs (siRNAs), which are then amplified by Rdp1. The siRNAs interact with Argonaute and guide chromatin remodeling complexes to the genome sites with sequences complementary to these siRNAs. Also occurring in the same regions are the histone H3K9 methylation by Clr4 and subsequent recruitment and oligomerization of the HP1/Swi6 protein, resulting in heterochromatin formation and spread in fission yeast (Grewal, 2010). The conserved features of fission yeast in chromatin organization and modification, especially the presence of RNAi pathways, reveal the unique value of S. pombe as a unicellular eukaryotic model to decipher mechanisms of epigenetic gene silencing.

Gene Structure and RNA Processing

In addition to the chromosomal organization characteristics discussed above, the structure of genes is another feature that S. pombe shares more in common with metazoans than with S. cerevisiae. Based on genome sequences, ~50% of fission yeast genes contain one or more introns. This corresponds to about 2,510 genes, of which most contain greater than two introns with 15 as the maximum intron number within one single gene (Wood et al., 2002; Wood et al., 2012). In comparison, only 5% of budding yeast genes harbor introns, accounting for about 344 genes and a total of 376 introns in its genome. The structural similarity of genes in S. pombe with those in complex eukaryotes is indicative of functional conservation in the gene expression process. Specifically, the relatively large number of introns, multi-introns within a gene, patterns of exon-skipping splicing, and the presence of SR-like proteins critical for alternative splicing in S. pombe, recapitulate the features of splicing regulation in metazoans as means of modulating gene expression. Similar to humans, splice sites in fission yeast display sequence degeneracy (Figure 8, (Fair & Pleiss, 2017). Also, evidence indicates that ~2-3% of all splicing events utilize alternative splice sites in S. pombe (Bitton et al., 2015). Moreover, analysis of exon-skipped, alternatively spliced transcripts in fission yeast, attributes such exon-skipping events to weak downstream 5′ splice sites (Stepankiw, Raghavan, Fogarty, Grimson, & Pleiss, 2015). Further, quantitative global transcriptomics data, acquired through a genome-wide screening assay using non-essential S. pombe gene deletion mutants, helped identify novel splicing factors, adding to the list of conserved SR proteins, including the SR-related gene pwi1 (SRRM1 human ortholog) and other known chromatin modifiers (Fair & Pleiss, 2017; Larson, Fair, & Pleiss, 2016).

Figure 8. Comparison of intron splice site consensus sequences in S. cerevisiae, S. pombe, and H. sapiens.

The height of each base directly correlates to the conservation of the nucleotide at that position. The nucleotide degeneracy at consensus splice sites is a feature shared between humans and fission yeast, while budding yeast displays relatively greater stringency at intron splice sites. Adapted based on (Fair & Pleiss, 2017).

To summarize, a distinctive advantage of using fission yeast lies in its suitability to serve as an experimental system to investigate RNA processing, a step of gene expression that is evolutionarily conserved in complex metazoans. While lacking in budding yeast, many mammalian-like features, such as degenerate splice site sequences and exonic splicing enhancers, are retained in the fission yeast. S. pombe also has a moderately complex spliceosome, undergoes regulated splicing with supporting SR-like proteins, and requires the splicing factor U2AF. S. pombe, therefore, is a promising model organism for investigating regulatory mechanisms of splicing and the consequences of misregulated splicing in human diseases (Sridharan, Heimiller, & Singh, 2011).

NOTABLE DISCOVERIES

The unique and most recognized discoveries made in S. pombe relate to understanding the molecular mechanisms of cell cycle regulation, which is fundamental to all eukaryotes through evolution (Fantes & Hoffman, 2016; Hoffman et al., 2015). In the 1980s, two seemingly distinct models for cell cycle regulation were proposed based on research in yeasts and early frog embryos: the domino model and the clock models, respectively. The domino model emphasizes the interdependence of the cell cycle events, whereas the clock model envisions a cytoplasmic timer controlling the onset of mitosis independent of other events in the cell cycle (Tang, 2010, 2016). These apparently different models reflected, at the time a knowledge gap in the principles of cell cycle regulation, mechanisms that are now known to be common to all eukaryotes.

Are the governing principles of the cell cycle conserved in different eukaryotic organisms through evolution? Is there any generality in cell-cycle regulation? Fission yeast as a model organism has greatly contributed to addressing these important questions. First, cell-division cycle (cdc) mutants were isolated in S. pombe, which uncovered the defined order of phase transitions in the cell cycle. Further, the genes responsible for the mutant phenotypes were then cloned to identify major regulators of the cell cycle control; Cdc2/Cdk1 master kinase, Cdc25 phosphatase, and Wee1 kinase. Finally, using cross-species complementation assays in S. pombe, the counterparts of those key regulators were found in humans (D. Beach, Durkacz, & Nurse, 1982; Lee & Nurse, 1987; Nurse et al., 1976; Russell & Nurse, 1986, 1987). The divergent views regarding cell cycle regulation between yeasts and animals were then converged at the molecular identification of the key regulators to demonstrate the conservation of cell cycle control through evolution from yeasts to humans.

Cell-division Cycle Mutants, Order of Phase Transitions, and the Domino Model

The early studies of the cdc mutants in both fission and budding yeasts revealed the order of the cell-cycle events and built the foundation for developing the concept of checkpoint control of cell cycle (Hartwell et al., 1974; Hartwell & Weinert, 1989). If a cdc mutant is blocked in S phase without completing DNA synthesis, it cannot initiate a later phase such as mitosis. This sequential order of the cell cycle events underlines the importance of checkpoint pathways, which are control mechanisms to ensure that phase transitions in the cell cycle occur only at a specific time and in a defined order. In essence, the initiation of a later cell cycle phase is dependent on the proper completion of a prior phase, known as the domino model of cell-cycle regulation — the central idea behind checkpoint control of the cell-division cycle (Hartwell & Weinert, 1989).

What can the fission yeast S. pombe offer as a model organism in the discovery pathways for cell cycle studies?

The biological nature of S. pombe allows for bridging genetics to cell biology. In particular, the regular cell shape, uniform cell size/length at division under defined conditions, and the highly polarized growth pattern of S. pombe have enabled scientists to isolate mutants defective in cell cycle progression based on the cell morphology (Table 1). Simply put, as cell length is reflective of a cell’s position in the S. pombe cell cycle (Figure 4A), cell length measurements provide a convenient and precise way to monitor the progression of the cell cycle. Therefore, S. pombe mutants with abnormal cell sizes, longer or shorter than that of wild-type cells, may be indicative of their defect in the cell cycle.

Based on this unique relationship between cell size and cell cycle stages, Paul Nurse began to search for mutations in S. pombe that would display abnormal cell sizes in the 1970s (Nurse et al., 1976). He used the forward genetics approach, which involved isolation of strains possessing chromosomal mutations that conferred a distinct cell-division cycle (cdc) phenotype, followed by identification of the gene alteration(s) responsible for the observed trait. Nurse and colleagues obtained more than 20 mutants corresponding to alterations in over 10 genes implicated in DNA synthesis, nuclear division, and septum formation. These cdc mutants were arrested at a distinct stage of the cell-division cycle after the incubation temperature was shifted from 25 °C to 35 °C, and thus were categorized as temperature-sensitive (ts) mutants. S. pombe cdc ts mutants usually display cell elongation, as the cell division is blocked at G₂/M, while growth continues at the restrictive temperature (Figure 9). Nurse and colleagues also observed another type of mutants, either null or ts mutants, which divided at a smaller cell size under corresponding conditions, exhibiting a wee (derived from a Scottish word for small) phenotype (Figure 9).

Figure 9. Typical phenotypes of temperature-sensitive mutants of cell cycle in S. pombe and S. cerevisiae.

The most common cdc phenotype in fission yeast is cell elongation, as a result of cell division block at G₂/M, while growth continues at the restrictive temperature. cdc2^ts or cdc25^ts are the examples provided. In contrast, a mutant CDC28 gene leads to an arrest at G₁/S with no budding formation at a restrictive temperature. A wee phenotype can be conferred by a defect in the gene product that normally inhibits passage through G₂/M transition without reaching a size requirement (wee1^ts). It can also be rendered by a gain-of-function mutation of a gene that normally promotes mitosis (cdc2-1w) at a restrictive temperature.

Why are cdc mutants commonly temperature sensitive?

Since cell division is essential for viability, if a haploid mutant harbors loss-of-function or null deletion mutation in a gene that is required for completing the cell cycle, it would be a lethal mutant. In a conditional mutant, a gene product behaves like a wild type under one condition but not another. Temperature changes are the most convenient conditions to turn a gene on or off. In a ts mutant, the gene product is as functional as in wild-type (wt) cells at lower, permissive temperatures, but not at higher, restrictive (or non-permissive) temperatures. Therefore, when examining a ts cdc mutant of S. pombe we can grow the cells at the permissive 25 °C temperature, then shift the temperature to 35 °C to observe the phenotype, thereby assessing the role of a gene product in the cell cycle. Most of the cdc mutants are ts, recessive, and loss-of-function mutations.

What are the features of cdc mutants?

At permissive temperatures, a cdc mutant of an asynchronous cell undergoes various stages of the cell cycle, similar to wt cells. The mutant cells become synchronous and arrest at the same stage of the cell cycle after the temperature is shifted to a restrictive temperature for 4-6 hours, irrespective of where they were in the cell cycle at the beginning of the switch (Figure 10). The specific point in the cell cycle, at which a cdc mutant is blocked under the restrictive conditions, depends on the function of the specific gene that has been mutated. If the specific gene product is required for cells to progress through the G₁/S, G₂/M, or metaphase/anaphase transitions, all the cells in the mutant population would be arrested at G₁, G₂, or M phase, respectively. Upon release from the cell cycle block by shifting down to a permissive temperature, the mutant cell population would then progress through the cell cycle synchronously. The genes altered in these mutants are called cdc genes, and they play specific roles at a particular phase transition of the cell cycle. In contrast, non-cdc mutants may be deficient in continuous processes such as ATP production, necessary for biosynthesis and growth throughout all phases; thus, these non-cdc mutants may halt at any stage of the cell cycle as soon as the ATP reservoir runs out (Figure 10).

Figure 10. Distinctions of cell-division cycle (cdc) mutants from non-cdc mutants.

Temperature-sensitive (ts) cdc mutant cells arrest at the same point in the cell cycle when an asynchronous population of cells is shifted to the restrictive temperature. The exact stage of the cell cycle that a cdc mutant is blocked under the restrictive conditions depends on the function of the specific gene that is mutated. In comparison, most non-cdc ts mutant cells arrest at random positions throughout the cell cycle at the restrictive temperature.

Several dozen cdc genes were originally identified in fission yeast and budding yeast by using the forward genetics approach (Hartwell et al., 1974; Hartwell et al., 1973; Nurse et al., 1976). Cells in a population of a particular cdc mutant would be blocked at the same phase of the cell cycle — displaying the same cdc phenotype. For example, at restrictive temperatures, cells with ts loss-of-function mutations in the S. cerevisiae CDC28 gene would arrest at the G₁/S without bud formation, while cells with mutations in the S. pombe cdc2 gene would not pass G₂/M, resulting in cell elongation (Figure 9). On the other hand, wee mutants may be defective in gene products that normally inhibit cells from progressing through the G₂/M transition before they attain a proper size. The first wee mutants were mapped to a single gene, wee1 (Nurse & Thuriaux, 1980). Alternatively, while loss-of-function mutations in cdc2 block mitosis leading to cell elongation, a gain-of-function mutation of the same gene would promote mitosis and give rise to a wee phenotype as well (Figure 9). It is thus conceivable that several wee mutations were mapped to the cdc2 locus. These analyses not only revealed the genetic relationship between the wee1⁺ and cdc2⁺ genes, but also further demonstrated the crucial role of the cdc2⁺ gene in the regulation of phase transition and the order of cell cycle events — the central idea of the domino model. Moreover, the inactivation of cdc genes by a change in conditions, usually temperature, offers an efficient way to generate cultures that are highly enriched for cells in one particular stage of the cell cycle (synchronization). Cell-division cycle mutants (Nasmyth & Nurse, 1981) have been, and remain, powerful tools in studying the cell cycle.

Identifying Key Players in Cell-Cycle Control: Cdc2, Cdc25, and Wee1

Using genetics approaches, pioneers in the field discovered a cast of vital players in control of the cell-cycle in fission yeast, including the genes encoding Cdc2 as the master cyclin-dependent kinase required for G₂/M transition, Wee1 tyrosine protein kinases as an inhibitor of Cdc2 kinase, and Cdc25 tyrosine phosphatase as an antagonist to Wee1 function (Figure 11) (D. Beach et al., 1982; Nurse et al., 1976; Russell & Nurse, 1986, 1987). Further, the characterizations of additional mutations leading to cell cycle defects established the basis for many subsequent studies on the regulation of S phase or DNA replication per se, the function of the septation initiation network (SIN) to couple the completion of mitosis to the start of cytokinesis, and the final cell separation through cytokinesis (Hoffman et al., 2015).

Figure 11. The functional relationship among important cell cycle regulators Cdc2, Cdc25, and Wee1.

Cdk1 as the master cyclin-dependent kinase consists of a Cdc2 catalytic subunit and a cyclin A/B regulatory subunit. The complex is required for G₂/M transitions. Its activity is regulated by antagonistic Wee1 tyrosine protein kinase and Cdc25 tyrosine phosphatase. Wee1 phosphorylates Cdc2 to inhibit its kinase activity to prevent cells from entering mitosis, while Cdc25 dephosphorylates Cdc2 to activate its kinase activity for cells to complete G₂/M transition.

Following the isolation of the cdc mutants using forward genetics, tetrad analysis and genetic mapping were used to define the loci of genes involved (Hartwell et al., 1973; Nurse et al., 1976). A major step forward in the cell cycle field was the cloning of the genes responsible for the cdc phenotypes to unravel the molecular nature of the encoded proteins. Genetic complementation assays were used to clone several cdc genes crucial for the cell-cycle regulation in fission yeast (D. Beach et al., 1982). In genetic complementation assays, a cdc gene can be identified and cloned by isolating a DNA fragment that can complement a ts cdc phenotype to restore cell cycle progression at the restrictive temperature. To clone the fission yeast cdc2⁺ gene encoding the master kinase Cdc2/Cdk1, a library of plasmids containing fission yeast DNA fragments was introduced into cdc2^ts cells at 25 °C, and the cells were then incubated at 35 °C (D. Beach et al., 1982). The vast majority of transformed cells received plasmids that did not contain the cdc2⁺ gene and thus failed to divide at the restrictive temperature. In contrast, only the mutant cells that up took the plasmid containing the cdc2⁺ gene were capable of restoring their G₂/M transition, thereby producing many cells to form colonies (Figure 12). The rescuing plasmids were then recovered from the proliferating colonies, amplified in bacteria, and used to determine the sequence of the gene. Cloning the cdc2⁺ gene allowed researchers to deduce the amino acid sequence of the Cdc2 protein, providing valuable clues about its putative molecular function as a protein kinase (Simanis & Nurse, 1986). In principle, any cdc gene can be cloned by employing the complementation assay, as long as the inactivation of the gene under a condition confers a strong cdc phenotype of cell cycle arrest.

Figure 12. Identifying and cloning genes by complementation assays.

The cloning of the cdc2⁺ gene is used as an example to illustrate the principle underlining the approach. A library of plasmids containing fission yeast DNA fragments is transformed into cdc2^ts cells at 25°C, and the cells are then incubated at 35°C. Each plasmid harboring one DNA fragment is received by one cell. Most of the cells intake a plasmid carrying a gene without a related function to cdc2⁺, resulting in gene arrest at G₂ and cell elongation. The cells thus cannot divide to form colonies on plates when incubated at the restrictive temperature. Only the small fraction of mutant cells transformed with the plasmid containing either the wild-type cdc2⁺ gene or a functional gene homolog, can rescue the cdc phenotype of the mutant and restore the cell cycle. The complementing plasmid can be recovered from these colonies formed on plates; subsequently, the plasmid can be isolated to determine the DNA sequence of the complementing gene, as well as predict the amino acid sequence of the encoded protein and its potential function based on the known DNA sequence.

Diverse Systems, Conserved Mechanisms

— “One Small Step for Yeast……One Giant Leap for Mankind”

The significance of genetic identification of key players in the fission yeast cell cycle was further manifested through discoveries of orthologs of these players in other eukaryotes. As the domino model and the clock model present very different themes of the cell cycle in yeasts and early frog embryos, respectively, they seemed to imply that distinct regulatory mechanisms of the cell cycle had evolved from yeasts to animals. Remarkably, finding the human functional homolog of the fission yeast Cdc2 culminated the discoveries in S. pombe and established the conservation of cell cycle control through evolution from yeasts to humans.

Are key regulators of the cell cycle in the two yeasts conserved?

Fission yeast S. pombe and budding yeast S. cerevisiae are distant cousins, as they diverged from each other approximately 350–400 million years ago (Hoffman et al., 2015; Sipiczki, 2000) (Figure 2). Based on initial studies, the two yeasts did not seem to share many similarities in the cell cycle or its underlying mechanisms. However, this view was dramatically altered after a successful complementation assay was performed across the two evolutionarily distant yeasts.

To isolate the potential ortholog of fission yeast cdc2⁺ gene in budding yeast, Beach and Durkacz in the Nurse lab extended the logic of complementation assay by cloning cdc genes in S. pombe to search for orthologs in S. cerevisiae. The only difference in the experiments was the use of a budding yeast gene bank (cross-species), instead of the fission yeast gene bank (in-species) to transform and rescue a cdc2 mutant in the complementation assay. They successfully identified S. cerevisiae CDC28⁺ as the functional homolog of the S. pombe cdc2⁺ gene (D. Beach et al., 1982). CDC28⁺ encodes a protein kinase homologous to Cdc2 kinase, initially known to be essential for G₁/S transition, and subsequently was determined to be necessary for the later stages of the cell cycle in budding yeast. Importantly, the CDC28⁺ gene could rescue the S. pombe cdc2 mutant and restore its cell cycle at the restrictive temperature. Therefore, the cross-species complementation assay provided a powerful strategy to study the cell cycle in an evolutionary context. It unraveled the conservation of the cell cycle control system — two diverging yeasts, one master player.

Are key regulators of the cell cycle conserved from fission yeast to humans?

Using a similar cross-species complementation assay, the human ortholog of the fission yeast cdc2⁺ was also identified, based on its ability to entirely replace the function of cdc2⁺ in S. pombe. All three genes—cdc2⁺ in fission yeast, CDC28⁺ in budding yeast, and CDC2⁺ in humans—not only play a similar function in the cell cycle but also encode closely related protein kinases with high sequence homology (Lee & Nurse, 1987). In 2001 for his milestone contributions to the cell cycle field using S. pombe as a model organism, Paul Nurse was recognized with the Nobel Prize in Physiology or Medicine, which was shared by Leland Hartwell and Tim Hunt.

Diverse biological systems, conserved mechanisms of cell cycle regulation — this is a milestone in the research field established on the basis of the discoveries in S. pombe. This finding has inspired us to further uncover the principles of cell cycle control fundamental to all eukaryotes. S. pombe has earned its reputation as a “micromammal”, holding promising potential and power as a unicellular eukaryotic model organism for understanding cell cycle and other biological processes in humans. “One small step for yeast……one giant leap for mankind” (Bussell, 2001).

ADVANTAGES AND ADVANCEMENTS

Although both S. pombe and S. cerevisiae are genetically tractable models, due to its closer phylogenetic distance than S. cerevisiae to humans (Figure 2), fission yeast offers unique advantages as a unicellular “micromammal” model for investigating the biological processes that have been lost in budding yeast through evolution (Forsburg & Rhind, 2006; Wood et al., 2002). Historically, classical genetic tools for tractable manipulation have only been available in yeast and Drosophila. Only recently, RNAi and CRISPR have been employed as gene expression and editing tools in mammalian systems, although these techniques are still primarily restricted to in vitro studies in cell culture settings. S. pombe, with its arsenal of tools ranging from classical and molecular genetics to cell biology, provides a powerful system for in vivo studies of different cellular processes. Here we focus on recent molecular genetic approaches developed in fission yeast, and their application to understanding the mechanisms of epigenetic gene silencing (White & Allshire, 2008), as examples of the strength of this model organism.

Molecular Genetics

The suitability of fission yeast for genetic manipulation has allowed for its extensive and successful use in classical genetics approaches to uncover several foundational and novel dogmas of cell biology (Forsburg, 2003; Tang & Edwalds-Gilbert, 2016). Its tractability in molecular genetics-based research further extends its efficacy as a robust tool for exploring gene function.

Inducible ectopic expression of various levels.

One of the widely used molecular approaches for investigating gene function is through vector-mediated ectopic expression of the gene of interest. One commonly used ectopic expression vector system in S. pombe is the pREP series, which provides a convenient method for manipulating gene expression at a desirable level for analysis. The plasmid vector pREP1 utilizes nmt1 (no message in thiamine), a thiamine repressible fission yeast promoter. The nmt1 is a very strong promoter; it shows a ~80 fold increase in expression level upon de-repression following thiamine removal (Maundrell, 1997). Targeted mutations in the TATA box of the original nmt1 have created two attenuated versions of this promoter; nmt41 and nmt81; with about 10 and 100 times lower expression levels than the nmt1 promoter (Basi, Schmid, & Maundrell, 1993). Thus, when transformed into fission yeast cells, the vectors pREP1, pREP41, and pREP81 carrying the corresponding attenuated promoters allow for a range of inducible expression levels for genes of interest under the control of promoter nmt1, nmt41, and nmt81, respectively (Figure 13).

Figure 13. Ectopic expression in S. pombe using pREP plasmid system.

The pREP plasmid system contains Leu2 selectable marker and autonomously replicating sequence (ars1) for plasmid amplification. Expression of a GFP-tagged gene of interest (goi) is under an nmt promoter. nmt1, nmt41, and nmt81 are related promoters with attenuated promoters of decreasingly weaker magnitudes. This system is thiamine repressible, and thus a GFP-tagged protein of interest (POI) is overexpressed in the absence of thiamine. In the case of a low endogenous expression level of a POI encoded by the corresponding goi, the system facilitates detection of the protein in the cells in order to examine its cellular localization and changes in post-translational modifications under different conditions. The system is also useful for determining the null phenotype of a goi by ectopically inducing or repressing the expression of the gene in a mutant strain, in which the endogenous gene is deleted.

The ability to vary gene expression levels based on the differences in promoter strength, and the ease of conditionally controlling gene expression through thiamine removal for de-repression at will, are very useful for exploring the function of genes in fission yeast cells. As an example, when the native expression of a gene of interest is low and difficult to detect in S. pombe cells, an ectopic expression system is used to boost expression. This is especially necessary to determine the cellular localization of the protein encoded by the gene of interest or examine its potential post-translational modifications for protein activity. The pREP vectors with promoters of different strengths may be selected to give rise to detectable levels of a gene product in vivo, while at the same time, avoiding overexpression leading to artifact observations. Additionally, a gene essential for viability may be deleted and replaced by an ectopic expression plasmid containing the gene. Similar to conditional mutants, using this ectopic expression vector system, genes can then be turned on for the growth of the mutant cells and then turned off to observe the effects of gene deletion, thereby providing insights into the normal gene function.

Based on the advances in S. pombe genomic sequencing, coupled with S. pombe’s innate ability for high precision of homologous recombination, new ectopic vectors have been designed that can stably integrate as single gene copies at specific genomic loci (Vjestica et al., 2020). These integration vectors contain a cassette harboring a gene of interest or a tag, and a specific selectable marker (ura4⁺, ade6⁺, his5⁺ or lys3⁺), flanked by sequences homologous to the genomic locus of that marker. Upon linearization, these vectors can be inserted at the specific locus of the auxotrophic allele (i.e. ura4⁻, ade6⁻, his5⁻ or lys3⁻), thereby converting a previously auxotrophic strain to prototrophic. The shared flanking regions of homology among a set of cassettes, allow for switching among cassettes containing different genes or tags at the same genomic locus. Simple sub-cloning using the appropriate cassettes with these vectors enhances their versatility, as it facilitates convenient introduction of additional selection markers and replacement with different genes or fluorescent tags (Vjestica et al., 2020).

PCR (polymerase chain reaction)-based gene targeting and tagging.

Following the availability of the fully sequenced S. pombe genome in the early 2000s, rapid advancements in fission yeast genetic tools ensued. This enriched the database of genetic information extensively, increasing a well-curated list of documented genetic, physical, and environmental interactions of genes and their gene products (Wood et al., 2012). In the post-genomics era, the in vivo gene manipulation technology in fission yeast has advanced at an unparalleled pace. For example, genes can now be tagged or disrupted directly at their endogenous genomic loci in S. pombe, using the so-called “one step PCR-based gene targeting” (Bahler et al., 1998). Briefly, PCR-amplified fragments containing DNA sequences encoding desirable factors such as mutant proteins, selectable markers, or green fluorescence protein (GFP) can be integrated into a specific genomic position through homologous recombination (Figure 14). The efficiency and accuracy of the integration rely on the correct design and generation of the PCR fragment with 100 bp-300 bp sequences homologous to those flanking the desired insertion sites (Figure 14) (Forsburg & Rhind, 2006). The advancement in gene targeting approaches has greatly facilitated studies on native protein expression and regulation, endogenous protein localization, modifications, and innate protein function.

Figure 14. Strategies of PCR (polymerase chain reaction)-based gene targeting and tagging in S. pombe genomic loci.

A. Overview of PCR-based gene tagging a gene of interest (goi) at the 3’ of the coding sequence. PCR primers on both sides contain at least 100 nucleotide sequences (nts) homologous to insertion locus at the 3’ of the goi, plus 20 nts from the plasmid flanking the gene encoding a tag to be amplified and inserted into the desired site. The nested primer method shown, circumvents the need for a primer longer than 100 nt. Instead of a primer of 120 nt, a forward pair and reverse pair, each consists of two 70 nt oligos with a 20 nt overlap to cover a total length of 120 nt. In addition to a gene tag, the PCR-amplified DNA fragment also harbors a selectable marker gene such as kanMX6, allowing for the selection of transformed colonies. The PCR-generated DNA fragment with 100 bp flanking sequences homologous to the genomic insertion site ensures its site-specific integration into the locus to express a C-terminal tagged fusion protein. B. Overview of PCR-based gene deletion. The strategy is the same as in A; however, PCR primers of forward and reverse contain at least 100 nts homologous to insertion locus at the 5’ and 3’ of a goi genomic locus, respectively, flanking the insertion site for a targeted gene deletion instead of gene tagging. As a result, a selectable marker such as kanMX6 is inserted in the locus of the gene to be deleted. Mutant strains with a stable integration of the PCR-fragment into the genome can then be selected by growing cells in an appropriate medium.

Epigenetic Gene Silencing

We will not an attempt a complete review on the advances in investigative methodologies using S. pombe, but the impact of this system on the field of epigenetic regulation and heterochromatin research cannot be left out of our discussion. As explained above, fission yeast and metazoans share conserved features in chromatin organization and modification and epigenetic regulatory functions. Especially the RNAi machinery, a conserved metazoan element, has been found in S. pombe, but has not been detected in budding yeast yet. Therefore, S. pombe offers a distinctive advantage as a model organism to decipher mechanisms of heterochromatin formation, maintenance, and spreading that govern epigenetic gene silencing (Cam & Whitehall, 2016).

Pericentromeric reporter gene silencing.

A clever genetic assay developed to study epigenetic gene silencing was constructed by inserting reporter genes within different heterochromatic regions of the centromere of S. pombe chromosome 1 (Allshire & Ekwall, 2015). The centromere 1 system has been used widely to investigate regulatory factors affecting chromatin state (Shimada et al., 2009). In this assay system, phenotypic observations reflect the switch between heterochromatin and euchromatin states, thereby allowing functional identification of factors involved in the regulatory circuit for gene silencing in centromere-associated heterochromatin regions (Figure 15). The adenine gene inserted at the otr1 region (outer repeat of centromere 1, cen::ade6⁺) containing the dh and dg repetitive sequences, acts as a transcriptional tracking tool (Figure 15A). If heterochromatin forms near the centromere in the outer repeat centromeric regions, the ade6⁺ expression would be transcriptionally silenced, conferring a red colony color phenotype when grown on a low adenine medium (Figure 15B, panel 2), similar to the control strain auxotrophic for adenine (panel 1). Alternatively, if the inhibition of the ade6⁺ expression from the otr1 region is partially lifted under the same condition due to changes in the centromeric chromatin structure, the colony color would display lighter red (Figure 15B, panel 3; unpublished data from Tang lab) or a progressive reduction in red color, compared with the positive control of ade6⁺ expression (panel 4). This colony color assay provides a color gradient-based or semiquantitative assessment of gene silencing in the pericentromeric region.

Figure 15. S. pombe centromere 1 system for pericentromeric reporter gene silencing.

A. The inserted ade6⁺ and ura4⁺ elements in the outer repeat (otr) and innermost (imr) regions of the centromere 1, respectively, serve as indicators of gene silencing/unsilencing from these domains. B. Colony color assay to monitor ade6⁺ reporter gene expression in the otr of the pericentromeric regions. Strain 501 (ade6-null) is a negative control of ade6⁺ gene expression. Strains grown on a plate with medium deficiency in adenine form red colonies, as they lack a functional ade6⁺ gene to produce adenine and instead produce a red intermediate (panels 1 and 2); while strains such as 972 expressing ade6⁺ form white colonies (panel 4). Mutation in a strain that changes the heterochromatin structure in the otr may unsilence or partially unsilence the ade6⁺ reporter gene, allowing for ade6⁺ gene expression and resulting in lighter red colored colonies (panel 3) C. Colony lethality assay to monitor ura4⁺ report gene expression in the imr of the pericentromeric regions. As 5-FOA is toxic to cells capable of producing uracil, strain 501 (ura4-null) serves as a negative control for ura4⁺ expression, thus is viable in the presence of 5-FOA, similar to the strain with the silenced ura4⁺ report gene in the imr domain (panels 1 and 2, bottom). In contrast, strain 972, as a positive control for ura4⁺ expression, displays lethality when grown in the medium with 5-FOA (panel 4, bottom). The viability is affected under the same condition for a strain with partially unsilenced expression of the ura4⁺ reporter gene in the pericentromeric region imr (panel 3, bottom).

Insertion of the ura4⁺ reporter gene into the innermost repeat 1 (imr1) region of centromere 1 (Figure 15A) offers a similar measure for heterochromatic gene silencing, as the colony color assay, although it probes the heterochromatin state of a different region flanking the centromere core, imr1, instead of otr1 (Allshire & Ekwall, 2015). This assay is based on the lethality of ura4⁺ strains due to the toxicity of 5-fluoroorotic acid (5-FOA) to cells capable of producing uracil, as shown in the control (Figure 15C, panel 4, bottom; unpublished data from Tang lab). When grown in presence of 5-FOA, a complete or partial loss of repression—or in other words, the expression of the ura4⁺ gene—results in lethality or reduced viability of the strain (Figure 15C, panel 3, bottom; unpublished data from Tang lab), compared with a strain auxotrophic for uracil (panel 1, bottom). However, in strains with effective maintenance and spread of centromeric heterochromatin, the imr::ura4⁺ transgene is silenced, and the strains grow well even in presence of 5-FOA (Figure 15C, panel 2). To sum up, the two reporter genes, ade6⁺ and ura4⁺ inserted into imr1 and otr1 of the centromere 1, respectively, provide simple yet powerful tools to monitor heterochromatic gene silencing from different pericentromeric regions based on their distinct phenotypes, colony color, and cell viability/lethality. It is important to acknowledge though, that fully quantitative measurement of ade6⁺ and ura4⁺ reports requires determination of mRNAs transcribed from the genes in the centromere 1 system (Cam & Whitehall, 2016).

Epigenomics.

Adding to the S. pombe toolbox for studying epigenetic regulation of gene expression is the development of a method for isolating “epimutants” to identify genes with a role in modulating the epigenetic mechanisms. For example, a recent study using S. pombe helped uncover a novel mechanism of caffeine resistance through acquired epigenetic changes. Caffeine exposure of fission yeast strains induced epigenetic changes to convert a subset of genes from a euchromatin to a heterochromatin state. This resulted in previously caffeine-sensitive strains becoming caffeine resistant (Torres-Garcia et al., 2020). Intriguingly, the selective epigenetic silencing was identified as heritable (passed on through several rounds of cell division) and yet reversible. Removal of caffeine relieves the heterochromatin-induced silencing in the subsequent generations. The histone methylation-mediated chromatin condensation in the resulting ‘unstable’ epimutants could be reversed in the generation relieved of caffeine exposure by the counteracting demethylase enzyme Epi1. However, the heterochromatic changes and epigenetic silencing would become irreversibly heritable as ‘stable’ epimutants, in strains that had a deletion in the epi1 gene. These findings have unraveled a novel mechanism of heritable, transient phenotypic plasticity implemented through epigenetic reprogramming. This epigenetic plasticity provides a bet-hedging strategy to enhance cell adaptability to a changing environment. S. pombe has indeed emerged as an attractive model for studying epigenetic regulation at a genome-wide scale, referred to as epigenomics (Allshire & Ekwall, 2015).

A Model for Systems Biology

Historically and contemporarily, fission yeast is well known for its suitability in forward as well as reverse genetics to dissect functional elements of various cellular processes (Forsburg, 2003; Tang & Edwalds-Gilbert, 2016). Since its genome was sequenced in 2002 (Wood et al., 2002), S. pombe has also arisen as a favorable model for systems biology-based studies, to investigate gene-gene and gene-organism-environment interactions (Deshpande et al., 2009; Wang, 2017). The creation of the S. pombe haploid deletion library, consisting of deletion strains for each of the 3,400 non-essential pombe genes, has provided an impactful tool to aid genomic screening projects (Kim et al., 2010; Spirek et al., 2010). For example, the S. pombe genome deletion library of haploid strains has been systematically screened to investigate the effect of drugs on the cells, based on changes in growth and morphology phenotypes (Wang, 2017). The library has been widely used in studying a range of cellular processes, from identifying novel genes and pathways involved in the DNA damage checkpoint pathways to determining drug response mechanisms (Deshpande et al., 2009; Pan et al., 2012). A particular strength of using S. pombe in genomics and proteomics analyses lies in its evolutionary closeness to the complex eukaryotes including humans. Furthermore, comparative genomics between the two divergent yeasts, S. pombe and S. cerevisiae, has enabled researchers to efficiently uncover novel and conserved genetic networks involved in cell-environment interactions as shown in the work of phenol stress response (Alhoch et al., 2019), accomplished mainly by undergraduate students. Further, as stated earlier, quantitative global transcriptomics data, acquired through screening assays using deletion mutants of non-essential S. pombe genes, enabled researchers to identify novel splicing factors (Fair & Pleiss, 2017; Larson et al., 2016). S. pombe fission yeast has contributed to the fruitful systems biology field since the post-genomic era.

RESOURCES FOR USING S. POMBE AS YOUR MODEL ORGANISM

Online resources for the S. pombe Community

1. PomBase – A comprehensive database that provides constantly updated and curated information on every pombe gene and molecular and biological data related to that gene. (www.pombase.org)

2. www.pombe.net–University of Southern California website with links to protocols, lists of meetings, investigators, plasmids, and other information, created and maintained by Susan Forsburg’s laboratory: https://dornsife.usc.edu/pombenet/fission-yeast-cell-cycle/

3. Jürg Bähler’s laboratory resource to help design CRISPR-based experiments in fission yeast: bahlerlab.info/crispr4p

Strain Availability for screening

Genome-wide deletion strains from collective work derived from the Korean consortium (https://eng.bioneer.com/20-m-5030h-lt.html).

Papers and books for working with S. pombe in laboratory

1. The Basic Methods paper, detailing essential fission yeast laboratory methods (Forsburg & Rhind, 2006).

2. PCR-based gene tagging using homologous recombination (Bahler et al., 1998).

3. Protocol paper describing the Lithium acetate-based transformation in fission yeast (Rai, Atwood-Moore, & Levin, 2018).

4. Reference for current stable integration vectors used in pombe-based research (Vjestica et al., 2020).

5. Book – A. Nasim et al, 1989, Molecular Biology of the Fission Yeast, A Volume in Cell Biology (DOI 10.1016/C2009-0-02773-1).

6. Book – Richard Egel, 2004, The Molecular Biology of Schizosaccharomyces pombe, Genetics Genomics and Beyond (DOI 10.1007/978-3-662-10360-9).

7. Book – Fission Yeast: A Laboratory Manual; Edited by Iain Hagan, Antony M. Carr, Agnes Grallert, Paul Nurse, CSHL Press (ISBN 978-1-621820-82-6)