Cell-Type Specification in Saccharomycotina

The life cycle of budding yeasts (Figure 1) is primarily comprised of three cell types: haploids of two isogamous mating types, a and α, and a/α diploids (Herskowitz 1988; Madhani 2007). The two types of haploid are often called mating types because they describe mating behavior: mating occurs only between a cells and α cells. Mating-type switching is the process by which a haploid a cell can become a haploid α cell, by changing its genotype at the mating-type (MAT) locus from MATa to MATα, or vice versa. Although it is historically called mating-type switching, the process could also be called cell-type switching.

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Figure 1

Schematic life cycle of S. cerevisiae.

All three cell types can divide mitotically given favorable environmental conditions, but, in S. cerevisiae, vegetatively growing haploid cells of opposite mating types will mate readily if they meet (Merlini et al. 2013). Haploid a cells express the G protein-coupled receptor Ste2, which detects the α-factor mating pheromone expressed by haploid α cells. Reciprocally, haploid α cells express the receptor Ste3, which binds the a-factor pheromone expressed by haploid a cells. Interaction between a pheromone and its receptor in either haploid cell type triggers a MAP-kinase signaling cascade resulting in G₁-phase arrest of mitotic proliferation, formation of a mating projection (shmoo) polarized toward the pheromone source, and finally mating by cell and nuclear fusion to generate a diploid zygote (Bardwell 2005; Jones and Bennett 2011; Merlini et al. 2013). Diploids are induced to undergo meiosis and sporulation by nutrient-limiting conditions in the environment (specifically starvation for nitrogen in the presence of a nonfermentable carbon source), resulting in the formation of an ascus. The ascus normally contains four haploid spores (two a’s and two α’s) that germinate upon restoration of favorable conditions (Honigberg and Purnapatre 2003; Piekarska et al. 2010; Neiman 2011).

Species within the fungal phylum Ascomycota vary as to whether they prefer to grow vegetatively as haploids (“haplontic”) or as diploids (“diplontic”) (Phaff et al. 1966). Whereas natural isolates of S. cerevisiae are primarily diploid, many other yeast species are primarily haploid, including K. lactis, S. pombe, and the methylotrophic yeasts such as Ogataea (Hansenula) polymorpha (Dujon 2010). Consistent with these ploidy preferences, S. cerevisiae mates spontaneously (even in rich media) and uses an environmental cue only for sporulation. In contrast, in haplontic yeasts, mating and sporulation are co-induced by poor environments and usually occur in succession without intervening diploid mitotic cell divisions, for example in Zygosaccharomyces, Kluyveromyces, Ogataea, Clavispora, and Schizosaccharomyces (Herman and Roman 1966; Gleeson and Sudbery 1988; Booth et al. 2010; Merlini et al. 2013; Sherwood et al. 2014). Sporulation of the zygote immediately after nuclear fusion results in a characteristic “dumbbell-shaped” ascus that retains the outline of the two shmooing haploid cells that formed it (Kurtzman et al. 2011).

The MAT locus controls processes dictating cell-type identity (Herskowitz 1989; Johnson 1995). For haploid cells (both a and α), cell type-specific processes include the induction of competence to mate and the repression of sporulation, whereas diploid cells require repression of mating and the ability to initiate meiosis and sporulation. Other processes also differ between haploid and diploid cells, such as the choice of location for formation of the next bud (axial vs. bipolar patterns; Chant and Pringle 1995), and the preferred mechanism for double-strand DNA (dsDNA) break repair (homologous recombination in diploids vs. nonhomologous end joining in haploids; Kegel et al. 2001; Valencia et al. 2001).

The MATa and MATα alleles (sometimes called idiomorphs) of the MAT locus are completely dissimilar in sequence. In S. cerevisiae the MATα allele contains two genes, MATα1 and MATα2, and the MATa allele contains a single gene, MATa1 (Figure 2). These three genes code for transcription regulators. They determine the cell type of the haploid by activating or repressing expression of a-specific (asg) and α-specific (αsg) genes (Johnson 1995; Galgoczy et al. 2004; Haber 2012). There are ∼5–12 asg’s and αsg’s, depending on the species (Sorrells et al. 2015). In addition to these, a shared set of haploid-specific genes (hsg’s) (∼12–16 in number) that facilitate mating is constitutively expressed in both a and α cells but not in a/α diploids (Booth et al. 2010), and a larger group of ∼100 general pheromone-activated genes is induced in haploids of both types once a pheromone signal from the opposite type of haploid is detected (Sorrells et al. 2015). For example, in S. cerevisiae, the pheromone genes MFA1 and MFα1 are an asg and an αsg, respectively; the pheromone signaling pathway G protein-subunit genes GPA1, STE4, and STE18 are hsg’s, and the MAP kinase FUS3 is a general pheromone-activated gene (Sorrells et al. 2015).

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Figure 2

Gene organization in the MAT, HML, and HMR loci on S. cerevisiae chromosome III. Shading indicates genes whose transcription is repressed.

In S. cerevisiae haploid α cells, the MATα1 gene codes for the HMG-domain transcription activator α1 (previously referred to as an “α-domain” protein but now recognized as a divergent HMG domain; Martin et al. 2010), and the MATα2 gene for the homeodomain-transcription repressor α2. The α1 and α2 proteins can both individually form complexes with the constitutively expressed Mcm1 (MADS domain) protein which binds upstream of asg’s and αsg’s. In α cells, transcription of αsg’s is activated because the α1-Mcm1 complex recruits the transcription factor Ste12 to their promoters, while transcription of asg’s is repressed because the α2-Mcm1 complex recruits the Tup1-Ssn6 corepressor (Figure 3).

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Figure 3

Logic of the cell type-specification circuits in Saccharomycetaceae species. Solid colors represent cell types (green, a; pink, α; brown, a/α), and outline colors represent gene sets. The genotype (1) of a cell’s MAT locus specifies the regulatory proteins present in that cell (2; checkboxes), which act at promoters (3) to generate appropriate transcription of the three gene sets (4; asg’s, αsg’s, and hsg’s) and determine the cell type (5). The yellow boxes describe the rewiring event that occurred when MATa2 was lost, coinciding with the WGD. The diagram summarizes information from the post-WGD species S. cerevisiae and the non-WGD species K. lactis, L. kluyveri, and C. albicans (Tsong et al. 2003, 2006; Booth et al. 2010; Baker et al. 2012; Sorrells et al. 2015).

In S. cerevisiae haploid a cells, the MAT locus contains only the MATa1 gene coding for the homeodomain protein a1, but this protein is not required for a cell-type identity. The identity of a cells is instead defined by the absence of both α1, the activator of αsg’s; and α2, the repressor of asg’s. Instead of requiring an a-specific activator, asg’s are activated by Mcm1 and Ste12, which are constitutively expressed in all cell types (Figure 3). Thus in S. cerevisiae, the a cell type is the default type, and yeast cells lacking a MAT locus will mate with haploid α cells.

In a/α diploid cells of S. cerevisiae, αsg’s, asg’s, and hsg’s are all repressed. These cells have MATα1 and MATα2 genes at the MAT locus on one chromosome, and MATa1 on the other (Figure 2), which results in formation of the a1-α2 heterodimer of the two homeodomain proteins. The a1-α2 dimer directly represses transcription of hsg’s, and indirectly suppresses αsg’s through repression of MATα1 (Figure 3). Transcription of asg’s in diploids is repressed by α2-Mcm1 as in haploid α cells. Because S. cerevisiae uses the formation of a heterodimer to sense heterozygosity of its MAT locus, and because this heterodimer is a repressor, there are no “diploid-specific” genes in S. cerevisiae (Galgoczy et al. 2004). Instead, diploid-specific processes such as meiosis and sporulation are repressed in haploids. This repression is achieved via the hsg RME1, a haploid-specific activator that transcribes IRT1, a noncoding RNA which in turn represses IME1, the master inducer of meiosis (van Werven et al. 2012). Thus the combined action of RME1 and IRT1 inverts the output of the hsg regulatory logic to restrict IME1 expression to diploids (Figure 3). IME1 expression also requires the environmental signals of nitrogen and glucose depletion that initiate meiosis (Neiman 2011). No genes have constitutive diploid (a/α) specific expression in the same way that hsg’s, αsg’s, and asg’s have constitutive cell type-specific expression in haploids.

The asg’s and αsg’s regulated by the MAT locus primarily include genes for pheromones, their receptors, and signaling proteins required for recognition of cells with the opposite mating type (Johnson 1995; Galgoczy et al. 2004). Binding of the pheromones to their receptors triggers a signaling cascade called the pheromone response pathway, which induces further gametic differentiation toward mating competence in haploids (Wittenberg and La Valle 2003; Merlini et al. 2013). This cascade culminates in the activation of the transcription factor Ste12, which is required for the expression of a large number of genes responsible for mating—the general pheromone-activated genes (Roberts et al. 2000; Sorrells et al. 2015). Ste12 is expressed in all cell types but when haploids detect a pheromone, Ste12 becomes substantially more active at pheromone-responsive promoters because the Fus3 MAP kinase inactivates two proteins, Dig1 and Dig2, that inhibit Ste12 (van Drogen et al. 2001). Ste12 is required for expression of all asg’s, αsg’s, and hsg’s. It binds to hsg promoters as a dimer that can be occluded by the a1-α2 complex in diploids, and it is brought to αsg promoters by α1-Mcm1. Ste12 also activates asg promoters in conjunction with Mcm1, although asg regulation has undergone recent dramatic evolutionary change (Sorrells et al. 2015).

Rewiring of the Logic Circuit After Whole-Genome Duplication in Saccharomycetaceae

The cell type-specification circuit of S. cerevisiae has undergone extensive reorganization since it diverged from other species in the yeast family Saccharomycetaceae, such as Candida albicans and K. lactis. The reorganization involved three distinct steps: the gain of α2 binding sites upstream of asg’s to repress them in α haploids (Tsong et al. 2006), the gain of Ste12 binding sites upstream of asg’s so that they are expressed by default (Sorrells et al. 2015), and the complete loss of the MATa2 gene. MATa2 codes for an HMG-domain transcription activator¹ called a2, which acts as an activator of asg’s in C. albicans and K. lactis (Tsong et al. 2003, 2006; Baker et al. 2012; Sorrells et al. 2015). Interestingly, the second and third of these steps occurred on the same branch of the phylogeny as the whole-genome duplication (WGD) (Sorrells et al. 2015), but whether they predated or postdated the WGD is not known. Recent phylogenomic analysis shows that the WGD was in fact an interspecies hybridization between two yeast lineages: one related to Zygosaccharomyces/Torulaspora, and one related to Kluyveromyces/Lachancea/Eremothecium (Marcet-Houben and Gabaldon 2015). MATa2 is present in most non-WGD species including these two parental lineages, but is absent in S. cerevisiae and all other post-WGD lineages including early diverging ones such as Vanderwaltozyma polyspora (Scannell et al. 2007; Wolfe et al. 2015).

Figure 3 summarizes the logic of the cell type-specification circuit in multiple yeast species, and how the output of this logic circuit remained unchanged by the rewiring event (Tsong et al. 2006; Sorrells et al. 2015). In species such as C. albicans and K. lactis that retain the MATa2 gene, repression of asg expression is not required in α cells, because asg’s are not on by default, so the repression of asg’s by α2 occurs only in S. cerevisiae. Therefore C. albicans MAT-deletion strains are sterile rather than defaulting to haploid a mating behavior (Tsong et al. 2003). The phylogenetic relationship (Figure 4) suggests that the cell specification circuit in C. albicans and K. lactis (Figure 3) represents the ancestral state of the network. Loss of a2 in the post-WGD lineage was only possible because, prior to this event, the promoters of asg’s in this lineage gained direct DNA-binding sites for Ste12 (Sorrells et al. 2015). In the ancestral situation, Ste12 was brought to asg promoters indirectly by a protein-protein interaction with a2, as occurs in K. lactis. An intermediate state survives in K. wickerhamii and Lachancea kluyveri, where some asg’s have “hybrid” promoters that are both repressed by α2 in α cells and activated by a2 in a cells (Baker et al. 2012). In addition to the loss of MATa2 in the post-WGD clade of Saccharomycetaceae, other variations in MAT gene content can be found in the CUG-Ser (Candida) clade, in which multiple species lack the homeodomain genes MATa1 and/or MATα2, and one species appears to have no MAT genes at all (Butler et al. 2009; Butler 2010).

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Figure 4

Phylogenetic tree of phylum Ascomycota showing major clades, MAT-locus organization, and known or inferred mating-type switching mechanisms. Based on Riley et al. (2016), with placement of A. rubescens as in Shen et al. (2016). Mating-type switching does not occur in species with only one MAT-like locus or in Aspergillus nidulans, which is a primary homothallic species.

Cell-type specification in yeasts is achieved by combinatorial regulatory gene presence or absence (Figure 3). The requirement for one copy of each MAT allele (MATa or MATα) to form the heterodimeric repressor acts as a sensor of ploidy, ensuring that only diploid cells are competent for meiotic entry (Haag 2007), and that diploid cells are incompetent for additional mating events that would result in aneuploidy. Furthermore, the presence of only one active MAT allele in haploid cells prevents the expression and recognition of self-pheromone that would otherwise induce mating processes in the absence of a mating partner. It has therefore been suggested that the structure of the MAT locus acts as a “developmental switch” that triggers mating and sporulation responses at the appropriate stages of the yeast life cycle (Perrin 2012).

Heterothallism and Two Types of Homothallism

Classical mycology makes a distinction between homothallic (self-fertile) and heterothallic (self-sterile) species of fungi. In homothallic species, any strain can mate with any other strain. In heterothallic species, strains fall into mating types (usually two of them) such that mating is only possible between strains of different types. Mating types are not the same as genders. For example, a single mycelium of the heterothallic filamentous ascomycete Podospora anserina can produce gametes with both male and female morphology, but each gamete is only able to fuse with a gamete that has the opposite morphology and that comes from a thallus (vegetative body) of the opposite mating type (Coppin et al. 1997).

The definition of homothallism becomes more finessed when applied to cells instead of strains, which is crucial for yeasts because they are unicellular organisms. At the cellular level there are two very different forms of homothallism: primary and secondary (Lin and Heitman 2007; Almeida et al. 2015; Inderbitzin and Turgeon 2015; Wilson et al. 2015). In primary homothallic species, any cell can mate with any other cell. That is, any two haploid cells can go through cell fusion and nuclear fusion to form a diploid. In contrast, in secondary homothallic species, mating occurs only between two cells with opposite mating types, just like in heterothallic species. However, cells of secondary homothallic species can switch their mating types quite easily. Consequently in these species, a strain, which is a population of cells that has been grown from a single progenitor cell, does not have a permanent mating type. Any strain can mate with any other strain because, even if the two progenitors originally had the same mating type, some cells in the cultures grown from them will switch their mating types, enabling them to mate with unswitched cells from the other strain. S. cerevisiae is a familiar example of a secondary homothallic species, although most laboratory strains and many natural isolates of this species have become heterothallic due to mutations in the HO gene (Mortimer 2000; Katz Ezov et al. 2010). Indeed, mating-type switching was one of the first discoveries in S. cerevisiae genetics because the isolates used by Carl Lindegren and Øjvind Winge in the 1930s were, respectively, mutant and wild type for this gene (Winge and Roberts 1949; Mortimer 2000).

Secondary homothallism by mating-type switching has been rigorously examined in Saccharomycotina (S. cerevisiae and its relatives) and Taphrinomycotina (S. pombe) (Klar 2007; Haber 2012; Klar et al. 2014; Lee and Haber 2015). In contrast, surprisingly little is known about the molecular basis of primary homothallism. In Pezizomycotina, primary homothallics such as some Cochliobolus species have a single type of MAT locus containing genes normally found in both alleles, whose structures indicate that they were formed by recombination between MAT chromosomes of heterothallic ancestors (Yun et al. 1999; Inderbitzin and Turgeon 2015). In Taphrinomycotina, it has recently been proposed that the pathogen Pneumocystis is a primary homothallic because it too has MAT genes from both alleles in close proximity on the same chromosome (Almeida et al. 2015). In Saccharomycotina, in our opinion, the only known strong candidates for primary homothallism are Debaryomyces hansenii and Scheffersomyces (Pichia) stipitis (Riley et al. 2016). They both have MATa1, MATa2, and MATα1 genes in proximity (Butler 2010), and meiotic recombination has been reported in S. stipitis crosses (Melake et al. 1996; Bajwa et al. 2010). However, the functions of their MAT genes and the details of what pheromone/receptor interactions occur in primary homothallic yeasts remain almost completely uninvestigated. Most fundamentally, it is unclear if and how cells of these yeasts avoid responding to their own pheromones. It has been suggested that primary homothallic filamentous fungi possibly sidestep this problem by expressing different pheromones and receptors in male and female tissues (Coppin et al. 1997; Martin et al. 2013), but this proposal has not been investigated experimentally and this solution is not available to unicellular yeasts (Billiard et al. 2012).

Multiple Mechanisms of Yeast Mating-type Switching

Programmed differentiation processes mediated by genomic DNA rearrangement are rare, with only a handful of examples known among all living organisms, and can affect single genes or entire genomes (Zufall et al. 2005). For organisms in which the germline genome is sequestered from the somatic genome, changes to somatic DNA can be irreversible and involve the loss of genes in differentiated tissues. For example, rearrangements during lymphocyte differentiation in vertebrates involve recombination between separate loci, resulting in antibody diversity and a robust immune response (Jung et al. 2006; Saha et al. 2010). Chromatin diminution during somatic tissue development in nematodes results in a more streamlined somatic genome by removal of heterochromatic regions (Muller et al. 1996; Muller and Tobler 2000). Ciliates, unicellular eukaryotes whose germline DNA is harbored in a micronucleus, dramatically fragment and amplify somatic DNA during development of the macronucleus (Chen et al. 2014). Unicellular organisms that do not contain separate germline and somatic DNA cannot make permanent changes to their genomes during development, as they will be transmitted to offspring. Instead, programmed DNA rearrangements underlying cell-type specification in these organisms must be reversible (Nieuwenhuis and Immler 2016). In addition to mating-type switching in yeasts, examples of reversible rearrangements include the shuffling of variant surface glycoprotein genes in kinetoplastids (Li 2015) and phase variation in Salmonella (Simon et al. 1980), both of which facilitate evasion of the host immune system.

Switching in S. cerevisiae involves unidirectional DNA replacement. The current gene content at the MAT locus of a haploid cell is replaced by copying a reserve version of the MAT genes of the opposite allele, stored at a transcriptionally silent location elsewhere in the genome (Haber 2012; Lee and Haber 2015). This process requires the genome to have three copies of mating-type sequence information, all of which are on chromosome III in S. cerevisiae: the active MAT locus (either MATa or MATα), two silent loci termed HML (containing the reserve copy of MATα sequence information), and HMR (containing the reserve copy of MATa sequence information). All three loci are flanked by identical sequence regions called X and Z (Figure 5). The Y region in the center comes in two forms, Ya and Yα, that are allelic but completely different in sequence. During switching, the actively expressed MAT locus is cleaved by the endonuclease HO. Guided by homology at the X and Z regions, the cleaved MAT locus uses HML or HMR as a template for DNA repair with a strong preference for the silent locus containing the mating-type information opposite to the current MAT genotype (Haber 2012). This mechanism of switching was named the cassette model (Hicks and Herskowitz 1977; Hicks et al. 1977) because the sequences at HML and HMR become inserted into the MAT locus for playback like cassette tapes in a player, and the three loci (MAT, HML, and HMR) are often described as cassettes.

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Figure 5

Organization of repeat sequences flanking the MAT loci in four species (Klar 2007; Hanson et al. 2014). In K. phaffii, the region that becomes inverted during mating-type switching is 138-kb long, and was recently discovered to contain a centromere at its approximate center (Coughlan et al. 2016). CEN, centromere; TEL, telomere.

Although mating-type switching in S. cerevisiae is often called gene conversion, it is more accurately described as a synthesis-dependent strand annealing (SDSA) process because of the nonhomology of the Y regions between the outgoing and incoming alleles (Ira et al. 2006). Switching is initiated when the HO endonuclease makes a dsDNA break at the Y-Z junction of MAT. The 3′ end of a DNA strand from the Z region beside MAT then invades the donor (HMR or HML) locus and is extended by DNA polymerase through the donor Y region and into the X region, after which it reinvades the MAT locus. Finally, the second strand of the new Y region at MAT is synthesized in the direction from X to Z. Switching is slow, taking ∼70 min, and is >1000 times more error prone than normal DNA replication (Hicks et al. 2010, 2011). Even though the newly synthesized MAT DNA will generally be replaced the next time the cell switches mating type, the high error rate nevertheless imposes an evolutionary cost because sometimes the errors will render the MAT genes nonfunctional.

Mating-type switching in the fission yeast S. pombe uses an analogous, but structurally unrelated, means to accomplish the same goal (Arcangioli and Thon 2004; Nielsen 2004; Egel 2005; Klar 2007; Klar et al. 2014). The S. pombe genome also contains one active (mat1) and two silenced (mat2 and mat3) mating-type loci (Figure 5). The proteins specifying the two mating types P (plus) and M (minus) include two HMG-domain transcription factors (Pc and Mc) and a homeodomain transcription factor (Pi), and thus share similarities with the S. cerevisiae proteins, but the mechanism of switching is different from that in S. cerevisiae. Instead of cleavage by an endonuclease, a fragile chromosomal site consisting of an epigenetic mark at the mat1 locus in switching-competent cells leads to a dsDNA break during replication. Repair of the break by SDSA using mat2 or mat3 as a donor is facilitated by homologous flanking regions, H1 and H2, which are analogous to the X and Z regions of S. cerevisiae but do not share sequence similarity or synteny with them (Beach and Klar 1984). The mechanisms used by S. pombe to silence expression of mat2 and mat3, and to bias the SDSA event to the locus containing the opposite mating type, are very different from those in S. cerevisiae (see below). The difference in mechanisms used to create the dsDNA break means that only one of the four mitotic grandchildren of a cell switches mating type in homothallic S. pombe strains, whereas two of the four switch in S. cerevisiae (Klar 2007; Hanson et al. 2014).

The lack of homology in every aspect of mating-type switching between S. cerevisiae and S. pombe has led to the remarkable conclusion that these distantly related species acquired their complex switching processes independently (Egel 2005; Lee et al. 2010; Rusche and Rine 2010). In this regard, it should be noted that S. cerevisiae and S. pombe underwent independent evolutionary transitions to unicellularity from a multicellular common ancestor (Nagy et al. 2014). Secondary homothallism by reversible changing of cell type is unknown in multicellular fungi and would not appear to serve any purpose in a multicellular context, so it is unlikely to have existed in the common ancestor of S. cerevisiae and S. pombe. A process known as mating-type switching has been described in some Pezizomycotina, in which half the progeny obtained by self-fertilization of a homothallic parent are heterothallic (Perkins 1987). This process has not been well characterized at the molecular level in any species, but it is consistent with models in which a recurrent inversion toggles between heterothallic and primary homothallic forms of a MAT locus (Chitrampalam et al. 2013), or in which a recurrent deletion converts a primary homothallic MAT locus into a heterothallic one (Witthuhn et al. 2000). There are no well-documented examples in Pezizomycotina of yeast-style secondary homothallism by a reversible switch between two mating types, each of which is self-sterile (Nieuwenhuis and Immler 2016).

In 2014, mating-type switching was reported in a clade of Saccharomycotina containing methylotrophic yeasts (family Pichiaceae; Figure 4) (Hanson et al. 2014; Maekawa and Kaneko 2014). These haploid yeasts contain one copy each of MATa and MATα loci, flanked by a pair of sequences forming an inverted repeat (IR) that are orthologous to the X or Z regions of the Saccharomycetaceae (Figure 5). One of the MAT loci is proximal to a heterochromatic region of the genome, a centromere in the case of O. polymorpha and a telomere in the case of Komagataella phaffii (these methylotrophic species are often called by their obsolete names H. polymorpha and P. pastoris, respectively). This arrangement confers repression of transcription on one MAT locus, while allowing active expression of the other locus, to specify cell type. Mating-type switching in these species occurs by recombination between the IRs, leading to inversion of the entire MAT region including all the genes located between MATa and MATα (19 kb in O. polymorpha, 138 kb in K. phaffii), swapping the positions of the active and repressed MAT loci relative to the centromere or telomere. It is important to note that switching in these species involves a reciprocal exchange of DNA: the previously expressed MAT genes become silenced, the previously silenced MAT genes become activated, and there is no new DNA synthesis. By contrast, in S. cerevisiae and S. pombe, the previously expressed MAT genes are degraded by exonucleases and replaced by newly synthesized DNA copied from the silent loci.

In recent work, we found evidence for similar flip-flop, mating-type switching mechanisms in two other yeasts (Riley et al. 2016). One is Pachysolen tannophilus, a haploid species in the methylotrophs clade (Figure 4). In response to nitrogen limitation, it inverts a 9-kb genomic region flanked by two identical sequences that form a 2-kb IR, similar to O. polymorpha. This 9-kb region has MATa1 and MATa2 genes at one end and MATα1 and MATα2 at the other end, and these loci are separated by ∼4 kb of noncoding DNA. While this process strongly resembles mating-type switching in O. polymorpha, its regulatory consequences have not been investigated. More significantly, a similar switching mechanism appears to operate in Ascoidea rubescens. Phylogenetically, A. rubescens was previously placed as deep lineage of Saccharomycotina that is an outgroup to all three major clades of this subphylum (Riley et al. 2016), although a recent phylogenomic study (Shen et al. 2016) placed it closer to family Saccharomycetaceae, as shown in Figure 4. Strains of A. rubescens were found to be polymorphic for the orientation of a 50-kb chromosomal region beside a telomere (Riley et al. 2016). Again, the invertible region is completely noncoding except for MATa1/a2 genes at one end and MATα1/α2 genes at the other, and the region is flanked by a 2-kb IR. Although it has not yet been shown that the orientation of the region can be induced to change, or that orientation affects expression of the MAT genes, the structure of the A. rubescens locus points to mating-type switching by inversion of a section of chromosome by recombination in the IR, placing either MATa or MATα genes beside the telomere.

The conservation of local gene order (synteny) around the MAT locus between the methylotrophs and the Saccharomycetaceae (Hanson et al. 2014) indicates that the two-locus inversion mechanism as seen in the methylotrophs and the three-locus (MAT, HML, and HMR) SDSA switching system as seen in S. cerevisiae share a common ancestor. It is likely that the two-locus system corresponds to a simpler ancestral mechanism of switching (Hanson et al. 2014), and this conclusion is supported by the existence of switching by inversion in both A. rubescens and the methylotrophs, regardless of which of the proposed phylogenetic positions of A. rubescens is correct (Riley et al. 2016; Shen et al. 2016).

More broadly, despite the variations in gene content, synteny around the MAT locus is reasonably well conserved among all ascomycetes. Although there have been many rearrangements in its vicinity, physical linkage of MAT to one or more neighboring genes (SLA2, SUI1, NVJ2, APC5, and APN2; none of which have known roles in mating) is widely conserved among the three subphyla: Saccharomycotina (budding yeasts), Pezizomycotina (filamentous ascomycetes), and Taphrinomycotina (fission yeasts) (Figure 4; Butler et al. 2004; Gordon et al. 2011; Riley et al. 2016). This conservation indicates that cell type has been specified by the same genetic locus throughout all of ascomycete evolution, even though there has been extensive turnover of the homeodomain and HMG-domain genes contained at MAT itself.

An ancestral mating-type switching system based on inversion of two MAT loci at least partly resolves the previously perplexing observation that two nonhomologous switching systems, both highly complex, appeared to have arisen independently and abruptly in the S. cerevisiae and S. pombe clades. We can hypothesize that a relatively simple ancestral inversion system has been made progressively more complex in the S. cerevisiae lineage through the addition of structural components and regulatory mechanisms, as discussed below.

Evolution of Mating-Type Switching Components

MAT-locus cassettes

The most obvious difference in complexity between the switching mechanisms of methylotrophs and those of S. cerevisiae and S. pombe is the number of MAT-locus copies each mechanism uses. The SDSA mechanism of S. cerevisiae and S. pombe requires a reserve copy of each MAT-locus allele as well as the active locus (three cassettes total), whereas the inversion mechanism of methylotrophs only requires one copy of each allele (two cassettes total). To guide the DNA recombination steps, the three-cassette system also requires two different sequences each to be present in triplicate (the X and Z regions; Figure 5), whereas a minimal two-cassette system requires only one sequence in duplicate (the IR) as seen in O. polymorpha, P. tannophilus, and A. rubescens. However, it must be noted that the methylotroph K. phaffii contains two sets of IRs in the MAT region and thus more closely resembles the X and Z structure of S. cerevisiae (Figure 5). In this species, mating-type switching occurs by recombination between the outer set of IRs. The function of its inner IRs is unclear, but exchange between them may act to restore collinearity between homologous chromosomes in diploids, enabling meiotic recombination to occur in the large (123-kb) interval between these IRs (Hanson et al. 2014).

In S. cerevisiae, four regions of sequence identity between MAT and the HM loci have traditionally been defined: W, X, Z1, and Z2 (Astell et al. 1981; Haber 2012), each a few hundred bp long. X and Z1 occur in three copies in the genome; whereas W and Z2 are regions that extend the similarity between MAT and HML, but not HMR, and so occur in two copies. In comparisons of other Saccharomycetaceae species, we found that in some cases the W and Z2 regions have negative length, i.e., the regions of similarity between MAT and HMR are longer than between MAT and HML (Gordon et al. 2011). Some species even have two HMR loci on different chromosomes, with different lengths of flanking sequence identity to MAT. We therefore do not think that W and Z2 have any functional significance separate from the roles of X and Z1, so for simplicity we use the names X and Z (instead of Z1) to refer to the triplicated regions, and ignore any extensions not shared by all the silent loci (Figure 5). We also usually draw the MAT locus in the order Z, Y, X, because in most Saccharomycetaceae species other than the genus Saccharomyces, the Z region is closest to HML and the telomere of the chromosome (Gordon et al. 2011). Saccharomyces sustained a bizarre trio of rearrangements, wherein MAT, HML, and HMR each became inverted by separate events (Fabre et al. 2005). The net effect of these three inversions, which are unique to the genus Saccharomyces, was to keep the orientations of HML, MAT, and HMR parallel, but with their X regions now closest to the left end of the chromosome.

Among Saccharomycetaceae species with the three-cassette system, the X and Z regions show very unusual evolutionary dynamics. To guide the DNA strand exchanges that occur during SDSA, the cell needs the sequences on each side of MAT to be identical to those beside HML and HMR, but the actual sequences that are triplicated vary enormously among species (Figure 6; Gordon et al. 2011; Wolfe et al. 2015). They usually consist of parts of some MAT genes and/or parts of the neighboring chromosomal genes. For example, in S. cerevisiae the X region contains the 3′ end of the MATα2 gene, and the Z region contains the 3′ end of the MATα1 gene. Switching from MATα to MATa replaces the 5′ ends of the two MATα genes (on Yα) with the whole MATa1 gene (on Ya), while switching from MATa to MATα does the opposite. Comparison among Saccharomycetaceae species reveals a remarkable diversity of ways that the X and Z repeats are organized relative to the four MAT genes (Figure 6). The primary evolutionary constraints on X and Z appear to be (1) to maintain homogeneity of the three copies so that DNA repair is efficient (they have a very low rate of nucleotide substitution; Kellis et al. 2003); and (2) to avoid containing any complete MAT genes within X or Z, so that the only intact genes at the MAT locus are ones that can be formed or destroyed by replacement of the Y region during switching.

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Figure 6

Seven ways to organize a MAT locus in family Saccharomycetaceae. The X and Z repeats, which occur in three copies in the genome (at MAT, HML, and HMR), overlap with parts of different MAT genes in different species. Each cartoon illustrates how the MAT genes are arranged, relative to the X and Z regions, in a group of species. The horizontal lines in each cartoon represent sequence that is shared between the MATα and MATa alleles, while the bubbles represent the divergence between the Yα and Ya regions. Blue arrows represent the neighboring chromosomal genes, which also vary among species (Gordon et al. 2011).

The diversity of organization of X and Z regions and their nonhomology among species is consistent with evidence that these regions have repeatedly been deleted and recreated during yeast evolution (Gordon et al. 2011). Comparative genomics shows that chromosomal DNA flanking the MAT locus has been progressively deleted during Saccharomycetaceae evolution, with the result that the chromosomal genes neighboring MAT differ among species. These progressive deletions have been attributed to recovery from occasional errors that occurred during attempted mating-type switching over evolutionary timescales (Gordon et al. 2011). Each time a deletion occurs, the X and Z regions need to be replaced, which must require retriplication (by copying MAT-flanking DNA to HML and HMR) to maintain the switching system. We only see the chromosomes that have successfully recovered from these accidents, because the others have gone extinct.

Evolution of Mating-Type Switching Regulation

Mating-type switching is inherently risky due to the need to make a double-strand break in a haploid genome. It is therefore tightly regulated both in direction, to ensure that it produces a cell of the required mating type, and in timing, to ensure that switching only occurs when it is most likely to result in successful mating. As discussed below, regulation is effected through multiple controls: on the choice of donor locus, by tracking the cell lineage and controlling the point in the cell cycle when switching occurs, and in some species by regulating switching in response to environmental conditions.

Resporulation as the Purpose of Mating-Type Switching

Although the mechanisms of mating-type switching have been explored in detail in several species, the evolutionary raison d’être of this process has received much less consideration. Switching is an error-prone process, as is clear from the elevated point-mutation rate at the newly synthesized MAT locus (Hicks et al. 2010) and the litany of gene deletions and transpositions that have occurred beside MAT during the evolution of the Saccharomycetaceae family (Gordon et al. 2011). From an evolutionary perspective, switching must therefore serve a function or provide a benefit to the host species that outweighs its mutagenic cost; otherwise natural selection would not have maintained the switching apparatus in so many species.

When thinking about the evolutionary advantage of mating-type switching, we need to disentangle the advantage of switching from the advantage of diploidy over haploidy as the primary mitotic growth form. Some yeast species that can switch mating types, such as K. lactis and O. polymorpha, are haplontic. Switching in these species normally leads immediately to mating and sporulation, without intervening diploid mitoses, so they appear to shun the often-cited advantages of a diploid mitotic lifecycle, such as the masking of recessive deleterious alleles or the ability to repair DNA by homologous recombination (Herskowitz 1988). S. cerevisiae is diplontic because it mates without requiring a nutritional signal, not because it can switch mating types. The phylogenetic distribution of diplonty vs. haplonty as the primary mitotic growth form of yeast species has not been studied in detail and is complicated by the existence of hybrids and species with cryptic sexual cycles (Dujon 2010). As a generalization, many post-WGD Saccharomycetaceae are diplontic and many outgroups to the WGD are haplontic (i.e., non-WGD Saccharomycetaceae, methylotrophs, and fully sexual CUG-Ser clade species), but there are several exceptions to this rule of thumb. For species that have the option of either haploid or diploid mitosis, the relative advantages of each may depend on the environmental context (Zorgo et al. 2013).

It could be argued that because mating-type switching increases the frequency of mating, the benefits of mating-type switching are the same as the benefits of sexual reproduction—that is, the ability to reassort beneficial alleles and purge deleterious ones (McDonald et al. 2016)—but this argument only applies if switching leads to outcrossing. The argument is consistent with the link between switching and transposable elements. Following spore germination, a diploid can be formed in one of three ways: by outcrossing (amphimixis), by intratetrad mating (automixis), or by haplo-selfing (mating between a mother and daughter cell after mating-type switching) (Greig and Leu 2009). Population genomic studies of S. paradoxus have led to estimates of the rates of these methods of diploidization in natural populations, with 94% of sexual generations resulting in intratetrad mating, 5% in haplo-selfing, and 1% in outcrossing (Tsai et al. 2008). Outcrossing has also been estimated to be as infrequent as 1 in every 50,000 generations in S. cerevisiae (Ruderfer et al. 2006), and the rate of outcrossing in S. cerevisiae is of the same order of magnitude as in L. kluyveri which cannot switch mating types (Friedrich et al. 2015). Thus mating-type switching appears not to be a significant factor contributing to the frequency of outcrossing in Saccharomyces.

Switching has the net effect of converting haploid cells into diploids. Yeast cells are not motile, so a haploid cell can only mate if it detects a haploid of the opposite type within shmooing distance. The effect of mating-type switching is that a haploid can create a mating partner for itself when no other potential partners are nearby. Several aspects of S. cerevisiae’s lifecycle indicate that the formation of diploids is a trait that has been strongly selected for in this species, leading to diplonty. First, in tetrad asci, the four haploid spores are arranged in a tetrahedral pyramid shape that ensures that every spore is positioned beside spores of the opposite mating type (Nasmyth 1982). Second, in circumstances where the sporulating diploid cell does not have sufficient resources to form a tetrad, the most common outcome is that a dyad (two-spored ascus) is formed instead, and a phenomenon termed spore number control results in the preferential production of dyads containing two nonsister spores that have compatible MAT loci to facilitate mating (Taxis et al. 2005; Lacefield and Ingolia 2006; Knight and Goddard 2016). Third, the axial budding pattern of haploid cells, in combination with S. cerevisiae’s restriction of mating-type switching to mother cells, ensures that when a spore germinates, MATa and MATα cells will be as close to one another as possible in the developing colony and therefore able to mate (Gimeno and Fink 1992). Fourth, asci of S. cerevisiae are “persistent,” meaning that the four spores are likely to remain together before germination (Knop 2006), and scanning electron microscopy of asci reveals interspore bridges that may promote intratetrad mating (Coluccio and Neiman 2004). Notably, these features of S. cerevisiae asci are not found in asci of haplontic species such as S. pombe, consistent with selection for features facilitating rapid diploidization in S. cerevisiae (Coluccio and Neiman 2004; Taxis et al. 2005; Knop 2006). Fifth and perhaps foremost, mating ability in S. cerevisiae is constitutive and does not require induction.

The maintenance of mating-type switching is simpler to rationalize in haplontic species that have fused sexual cycles with only transient diploid states. Cells that require an environmental signal to diploidize may grow for several haploid generations before mating conditions are encountered. This increases the probability that such a cell may not find a mating partner. The simultaneous induction of mating-type switching and mating would therefore be more likely to result in successful diploidization and subsequent sporulation. However, if a mating partner is present when mating is induced, mating-type switching will be unnecessary and should be repressed (such as by pheromone-signaling pathways) (Barsoum et al. 2011), although this hypothesis has not been tested in haplontic species.

What evolutionary pressures might act to maintain mating-type switching in diplontic species like S. cerevisiae? Mortimer proposed that it could be maintained because occasional haplo-selfing events are necessary to purge recessive deleterious alleles that accumulate during generations of asexual reproduction or intratetrad mating in the population (Mortimer et al. 1994; Mortimer 2000; Magwene 2014). This problem is exclusive to diplontic species, because in haplontic species the recessive alleles will be exposed during haploid mitotic growth. In fact, S. cerevisiae populations are polymorphic for the ability to switch, with null alleles of the HO locus being present in 25% of natural isolates in one study (Katz Ezov et al. 2010). Losses of switching can also be inferred to have occurred in two species in the family Saccharomycetaceae (L. kluyveri and Kazachstania africana) (Gordon et al. 2011), as well as in the large CUG-Ser clade of species related to C. albicans (Butler et al. 2009). One could therefore argue that although there has been evolutionary pressure to maintain switching in most yeast species, this pressure can sometimes disappear.

Our laboratory has proposed an alternative hypothesis, that switching is maintained by selection for the ability of a cell to resporulate after germinating (Gordon et al. 2011; Hanson et al. 2014). This hypothesis can be applied to both diplontic and haplontic species, and is based on the concept of a “lonely spore,” first proposed by Herskowitz (1988). A lonely spore is one that is physically isolated from any other spores of the same species, so that when it germinates it will give rise to a haploid colony unless it is capable of mating-type switching, in which case some cells in the colony will be diploid. The factors determining when a spore germinates are not well understood, but it seems likely that the control of germination timing is under strong selection. Spores are survival structures that can protect a cell during times when vegetative growth is not possible. In environmental conditions that are improving (changing from inhospitable to hospitable), a spore that germinates earlier than its peers will have a competitive advantage. However, if it germinates too early, the environment may not be sufficient and it could die. Because only diploid cells can form spores, we have argued that a mating-type switching system enables spores to try germinating earlier (to test the environment); because if the environment is inhospitable they can form new spores after as few as two rounds of cell division. In contrast, germination is an all-or-nothing commitment for a yeast strain that cannot switch mating types, because once it germinates it cannot make new spores unless it finds a mating partner. By computer simulation, we have shown that features such as donor bias and lineage tracking increase the proportion of diploid cells (or haploid cells with a potential mating partner) in the microcolony formed from a germinating spore, so the emergence of these features can be explained by natural selection for an increased ability of germinating cells to resporulate (Hanson et al. 2014). The change from a two-cassette to a three-cassette switching system can also be explained by this model, because donor bias is only possible in a three-cassette system.

If the purpose of mating-type switching systems is to maximize the ability of microcolonies to form new spores, the benefit of switching could be a direct one related to cell survival as envisaged above, or an indirect one related to cell dispersal. S. cerevisiae is not carried by wind and instead relies on vectors such as insects for its introduction into new environments (Mortimer and Polsinelli 1999; Goddard et al. 2010; Stefanini et al. 2012). The spore wall enables spores to survive digestion by Drosophila melanogaster (Coluccio et al. 2008). Thus, for S. cerevisiae, the induction of sporulation by a poor environment may be a means to disperse into a new and perhaps more favorable environment after meiotic recombination and the generation of potentially advantageous genotypic variation (Neiman 2011). The passage of spores through the gut of D. melanogaster has also been shown to increase the rate of outcrossing in S. cerevisiae (Reuter et al. 2007), suggesting that spores in asci become separated from one another during dispersal. Whether this spore separation also results in an increased rate of haplo-selfing is not known.

Mating-type switching can be viewed as a form of reproductive assurance (Nieuwenhuis and Immler 2016): it enables haploid cells to mate, even if they are immotile and dispersed at low density in the environment. Secondary homothallism by switching thus guarantees that most cells can mate, without losing the benefits of having separate mating types (such as increased genetic diversity through outcrossing) that are absent in primary homothallism. While we agree with this view, we would suggest that in yeasts, which have the option of long-term mitotic reproduction without sex, the purpose of assuring mating is not to assure reproduction but to assure that spores can be formed.

Perspectives

Recent advances (Hanson et al. 2014; Maekawa and Kaneko 2014; Riley et al. 2016) allow us to postulate that the ancestral mating-type switching system in the fungal subphylum Saccharomycotina consisted of a two-cassette, flip-flop system, which later developed into the more complex three-cassette SDSA system of family Saccharomycetaceae. The ancestral flip-flop system originated prior to the divergence between A. rubescens, the methylotrophs, and the Saccharomycetaceae (Figure 4). We do not know what mechanism was used to repress transcription of the silent MAT locus in this ancestor, but it cannot have been either the H3K9me2/3 system (which was already lost) or the SIR system (which had not yet been invented), so centromeric or telomeric silencing seems likely. One possible, albeit speculative, way that the first two-locus system might have originated is that a diploid cell of a heterothallic species could have sustained a chromosomal rearrangement that moved one of the MAT alleles onto the same chromosome as the other allele, but in a heterochromatic region (centromeric or telomeric). Recombination between repetitive elements such as transposons could then have allowed flip-flop switching to emerge, inverting the region containing the two MAT alleles.

One of the most intriguing aspects of mating-type switching is the extent of parallels between the switching systems of subphyla Taphrinomycotina (S. pombe) and Saccharomycotina (S. cerevisiae, K. lactis, the methylotrophs, and A. rubescens). Since these subphyla share a multicellular common ancestor, and mating-type switching does not occur in multicellular fungi, it appears beyond doubt that the Saccharomycotina and Taphrinomycotina switching systems are a dramatic example of convergent genomic evolution in which each system independently developed silent cassettes and donor bias. At present we have little information about what the ancestors of the S. pombe system looked like. The unicellular (yeast) lifestyle emerged from multicellular ancestors on at least five separate occasions during fungal evolution (Nagy et al. 2014). Taphrinomycotina and Saccharomycotina are two of these clades, but we can also wonder whether switching may have emerged in the other three clades, or why it did not. These three clades are all in Basidiomycota. One, which includes the genus Cryptococcus, has been studied extensively and shows heterothallism with no evidence of switching, but the others which include the genera Sporobolomyces and Malessezia are much less investigated.

A final more philosophical question concerns why cell-type specification in secondary homothallic yeasts like S. cerevisiae includes a DNA-rearrangement mechanism at all. Cell differentiation in most other eukaryotes is achieved using normal transcription factor networks, so why do yeasts use a chromosomal rearrangement to change mating types? It is interesting to compare the MAT system to the white/opaque switching system of C. albicans. This species has two yeast cell morphologies that are specified by the two stable states of a gene regulatory circuit with feed-forward loops (Zordan et al. 2007). In the opaque state, the Wor1 transcription factor is active and Efg1 is repressed; while in the white state, Efg1 is active and Wor1 is repressed; but the DNA content of white and opaque cells is identical. Switching between the two states occurs at a frequency of 5–16% (Zordan et al. 2007). Why can yeast mating type not be controlled by a simple network like this? The answer is that the states of a regulatory network cannot segregate in meiosis. If cell type were specified by the state of a network, a network in the a/α state would need to be able to go through meiosis and produce spores with only a or α networks, which seems impossible. Cell type needs to have Mendelian inheritance, so it needs to be controlled by alleles of a chromosomal locus. Therefore, in turn, if haploids are to switch mating types, they must be able to alter the DNA of that locus to exchange the alleles in a reversible manner.