Mating type tutorial – Department of Biology - University of Copenhagen

Cell cycle > Mating type tutorial

A Mating-Type Tutorial on Fission Yeast

Figure 1. The life cycle of Schizosaccharomyces pombe.

Vegetative cells are predominantly haploid (1n). Homothallic switch of mating type (P « M) occurs during vegetative growth. Zygote formation and/or meiosis occur after nutritional depletion, especially for a nitrogen source. Conjugation requires partner cells of opposite mating type, whilst meiosis requires expression of opposite mating-type genes in the same cell. Meiosis is followed by ascospore formation. Rarely occurring diploid growth of zygotes (2n) can be selected for in the laboratory. As homothallic mating-type switching is not suppressed in diploid cells, this can lead to further variations, such as conjugation of diploid cells and tetraploid meiosis (not shown).

Figure 2. Mating-type activities during conjugation and meiosis.

Either mating type is specified by two genes, which are tightly linked in a so-called mat-cassette: M by mat-Mc and mat-Mm (a.k.a. Mi); P by mat-Pc and mat-Pm (a.k.a. Pi). These genes cannot be expressed at all in G2, which is the predominant phase in actively growing cells of fission yeast. When G1 is extended upon nitrogen depletion, only Mc and Pc genes can be expressed initially. (A) If Mc is expressed in M cells, additional genes are being activated, encoding M-specific proteins, such as M-factor pheromone, M-cell agglutinin and a receptor for the opposite pheromone. (B) If Pc is expressed in P cells, the gene encoding the receptor for the opposite pheromone is expressed primarily. (C) In P cells the pheromone response has be activated by binding of M-factor at the Mf-receptor before additional genes can be induced that code for P-factor pheromone and P-cell agglutinin. Also, the activated P cell extends a conspicuous conjugation bulge in the direction of an M-factor gradient. (D) The binding of P-factor to Pf-receptor will likewise activate the pheromone response in M cells, which in turn form less conspicuous conjugation bulges. Together, activated M and P cells assemble in clumps by way of the complementary agglutinins, so as to facilitate the pairwise association of potential mating partners. (E) Other, pheromone-induced activities (not encoded by mating-type genes) result in cell fusion and karyogamy. Notably, the Mm and Pm mating-type genes are themselves induced by the pheromone response, but the corresponding proteins can only form active heterodimers after zygote formation, which then triggers the cascade of further reactions that leads to meiosis and sporulation (F).

Figure 3. Cassette organization in the mat region of S. pombe - homothallic wild-type.

(A) One expressible and two silent mating-type cassettes are carried as direct repeats on the right arm of chromosome 2. Their overall arrangement is indicated (not drawn to scale). In any given cell, the expressible mat1 cassette can be either P or M, whereas the silent mat2-P and mat3-M cassettes are fixed. The spacer regions are designated L and K, of which only the latter is devoid of other protein-coding genes. (B) Each mat cassette carries two separate genes, the functions of which are mentioned obove (Fig. 2). Furthermore, each cassette is flanked by homology boxes H1 and H2, important for the switching mechanism. (C) The mechanism of homothallic mating-type switching depends on several cis-acting elements at mat1. An imprintable site (!) marks the internal border of H1, where one of the strands can be labilized at 1-2 thymine residues - conceivably by oxidative conversion to ribonucleotides, or by the addition of reactive groups to the base moieties. The biochemical mechanism of the imprinting reaction is still unresolved. Sequence cues to specify the imprintable site are scattered over a remarkable length of ~300 base pairs ({). Inasmuch as the conversion of an imprint into an effective switch of mating type is coupled to DNA replication approaching from outside H1, potentially interfering replication from the other side is blocked by a potent directional termination signal (RTS1) outside H2. (D) The region encompassing mat2 and mat3 cassettes is kept silent by heterochromatin formation. This process starts internally at several nucleation sites, such as cenH inside the K region - with substantial homology to pericentromeric repeats. Inadvertant spreading into surrounding gene-rich regions is prevented by structural boundaries at two inverted repeats (IR-L and IR-R). (E) At higher resolution, the local organization of mat2-P and mat3-M reveals additional cis-acting signals. The homology boxes H1 and H2 are identical to those carried at mat1, and this is where the switching reaction by repair synthesis is initiated and resolved, respectively (see below, Fig.6). The additional H3 boxes are only found at the silent cassettes, where they participate at the resolution step. Adjacent to H3, the additional silencing elements RE2 and RE3 supplement the cenH-directed mechanism of heterochromatization.

Figure 4. Heterothallic strains by rearrangement

(spacer regions not to scale). The wild-type configuration of the homothallic h90 strain (middle lane) can lead to h-S by deletion, or h+N by insertion, between homology boxes H1 and H2 of different mat cassettes. The h+N insertion results from aberrent switching events (see below, Fig. 6C). Silent cassettes are shaded; imprintable sites to initiate mating-type switches are indicated (!). Historically, the strain symbols H90, h+N and h-S read "Homothallic, yielding 90% sporulation", "heterothallic, Plus-Normal" and "heterothallic, Minus-Stable", to distinguish them from yet further variations (not shown here). Operationally, the imprintable sites carried in all three strains can act as recombinational hotspots, which can have undesirable side effects - particularly in working with diploid strains. More reliable heterothallic derivatives have been engineered by deleting the silent cassettes and/or the imprintable sites.

Figure 5. The cycle of homothallic mating-type switching in S. pombe.

Mitotic cells are passing through four stages of asymmetric cell division:

  Pu → Pu+Ps;  Ps → Ps+Mu;  Mu → Mu+Ms;  Ms → Ms+Pu.

When switchable cells divide (Ps or Ms), one of their respective daughters will have switched mating type. On depleted medium, this allows conjugation and ascospore formation with the unswitched sister cell. If unswitchable cells divide (Pu or Mu), both daughter cells retain the same mating type and and are not mutually inhibited by mating pheromones - even though one of these becomes competent of switching in the next division. This leads to a single switch in the subsequent quadruplet of cousin cells. In molecular terms, the unswitchable cells are not yet imprinted at mat1-H1, whereas the switchable cells bear the imprint.

Figure 6. Mating-type switching by recombinational repair.

The imprint at mat1 acts as a strand-specific site of calculated damage, where the upcoming replication fork is stalled and only can be rescued by recombinational repair, using an undamaged homologous strand as a template. This process is diagrammed for the imprinted mat1-P cassette, preferentially using mat3-M as the template. The opposite switch from an imprinted mat1-M cassette will proceed similarly, preferentially using mat2-P as the template. Notably, the imprints at mat1 do not become lethal in the absence of silent donor cassettes. Presumably, the stalled replication forks can also be rescued by the intact complementary strand at mat1, which is not affected by the imprint. (A) A replication fork approaching from the left is halted outside mat1 at RTS1. The leading strand approaching from the right is halted at the imprint (!). (B) Facilitated by various recombinational repair factors, the leading 3' end is liberated from H1 at mat1, swapping template with H1 at mat3-M (i). Repair synthesis pursues throughout mat3-M, to be terminated at the H2/H3 boundary (ii). Around the H2/H3 boundary, the single-stranded product can fold up in multiple hairpin structures, which probably facilitate the resolution steps. The processed 3' end reenters mat1 at H2 (iii), to be joined with the arrested lagging strand at RTS1. This strand is duplicated immediately thereafter (not shown), to complete the newly formed mat1-M cassette. The intact strand of the resident mat1-P cassette is duplicated as well, and imprinted anew (not shown), whereas the previously imprinted strand is degraded. (C) If switching of mat1-P to M fails to be terminated at the H2/H3 boundary of mat3-M, repair synthesis continues throughout the entire K region, only to be resolved at the H2/H3 boundary of mat2-P. This aberrant switching event results in the heterothallic h+N configuration. It is rare in wild-type cells, but occurs more frequently in various mutant strains, where the resolution steps are affected specifically.