Fig 1.
Mating-type switching in S. pombe.
(A) The mat1 locus is expressed and confers cell type. The silent loci mat2-P and mat3-M serve as donors of genetic information to interconvert the mat1-P and mat1-M alleles. They are located in a heterochromatic region bordered by the IR-L and IR-R repeats. The small H1 and H2 homology boxes are common to all cassettes. H3 boxes are specific to mat2-P and mat3-M. RTS1: Replication Termination Site 1; MPS1: mat1-Pausing Site 1; SAS: Switch Activation Site; IR-L or R: Inverted Repeat Left or Right; RE: Repressor Element; SRE: Swi2-dependent Recombination Enhancer; cenH: centromere Homology; Rep-Ori: origin of replication. (B) Mating-type switching occurs in a defined pattern depicted here, with P cells represented in blue and M cells in yellow. The subscripts ‘u’ and ‘s’ designate unswitchable and switchable cells, respectively. (C) Steps in mating-type switching. A DNA modification or ‘imprint’ at the mat1-H1 junction is symbolized by a red mark. Leading-strand synthesis incoming from the indicated origin of DNA replication proceeds through the mat1-H1 homology box and stops at this modification creating a one-ended double-strand break (DSB). The resulting free DNA end invades the H1 box of the silent cassette containing the information for the opposite mating-type, mat2-P in this example. D-loop formation permits extension of the DNA molecule at mat2-P, until the newly synthesized H2 sequence copied from mat2-P anneals back to the H2 box at mat1 to resolve recombination. Completion of switching further requires second-strand synthesis (of mat1-P in the case shown) and degradation of the unused mat1 template strand.
Table 1.
Mating-type switching related genes.
Fig 2.
Screen for mutants defective in donor choice.
(A) Summary of screen. Fluorescence microscopy, multiplex PCR for mat1 content and iodine staining were used as described in the text to identify mutants with a mat1 content biased towards mat1-P or mat1-M. Candidates were selected for differing by > 3 standard deviations from the mean in the fluorescence and multiplex PCR tests. Lists of mutants and values are presented in S2–S4 Tables. (B) Examples of cell-type ratios measured by fluorescence microscopy with the dual reporter system using YFP under control of the mfm3 M-specific promoter and CFP under control of the map2 P-specific promoter. P-to-M cell ratios were determined with an Opera high-throughput microscope (Perkin Elmer Inc.). % of cell population were calculated from cyan/(cyan + yellow) cell ratios. The unmutagenized strain PG4045 is shown as control (cntl). See S2 Fig for additional mutants. (C) Spore content was assayed by exposure of colonies to iodine vapor. The starch in spore walls is stained darkly by iodine. Mating-type switching mutants form light colonies. (D) Multiplex PCR was used to measure mat1 content with a P- and an M-specific primer combined with a mat1-specific primer. The P and M band intensities in each lane were used to calculate P/(P+M) and M/(P+M) ratios reported under the gel pictures and as bar graphs for P/(P+M). The line at 42.3% in the graph shows the lower limit accepted as normal mating-type switching.
Fig 3.
Classification of mutants obtained in the screen according to imprint formation and rearrangements.
Southern blot analysis of HindIII-digested DNA. The probe was made from a 10.4 kb mat1 HindIII fragment. The positions of HindIII sites and primers used for PCR in S3 Fig (red arrow: mat1-specific; yellow: M-specific and blue: P-specific) are indicated above the blots. Strain PG4045 shows the hybridization pattern of a wild-type h90 strain. Mutants were classified according to imprint level and occurrence of specific rearrangements. The 5.4 kb and 5.0 kb bands are products of the DSB that occurs at the imprinting site during DNA preparation; they are absent in the Class Ia mutant, swi3Δ. mat3:1 (8.2 kb) and mat1:2 (6.7 kb) result from the h+N rearrangement of the mating-type region. These fragments are present in Class II mutants. The 8.2 kb band in Class Ib mutants might originate from a mat3:1 extrachromosomal circular element, or from a duplication of the mating-type region resulting from unequal sister chromatid exchange between mat1 and mat3. mat2:1 (9.9 kb) might result from a circular minichromosome in the Bioneer swd2Δ mutant. ‘?’ marks a band of unknown origin.
Fig 4.
Determination of donor-choice preferences in mutants with the h09 mating-type region.
The donor loci in h09 strains are swapped from mat2-P mat3-M to mat2-M mat3-P. Changes in mat1 content caused by the Class Ib mutations identified here were estimated by multiplex PCR in the h09 background, from the band intensities shown in S4 Fig. Red bars represent means ± SD. Clr4, Raf1 and Rik1 are components of the H3K9 methyltransferase complex CLRC. Sir2 is a deacetylase. Swd1, Swd2 and Spf1 are components of H3K4 methyltransferase complex, Set1/Compass, Clr1, Clr2 and Clr3 are components of the deacetylase complex SHREC. One-way ANOVA was used to compare means of each sample to cntl, ***p < 0.001; ****p > 0.005.
Fig 5.
Analysis of mating-type switching directionality of Set1C mutants.
(A, B) Spore formation of mutants in each Set1C subunit was assayed by exposure of colonies to iodine vapor. h90 strains are shown in (A) and h09 stains are shown in (B). (C, D, E) Quantification of mat1 content estimated by multiplex PCR. The relative P band intensity in each lane (P/(P+M)) was calculated from the data shown in S5 Fig for the h90 strains shown in (C); from S5 Fig for the h09 strains shown in (D) and from S5 Fig for the strains with mutated SRE elements shown in (E-H). (E) 2×SRE2 strains (mat2-P-SRE2 mat3-M-SRE2) were derived from the set1+ strain TP126; (F) 2×SRE3 strains (mat2-P-SRE3 mat3-M-SRE3) from TP303; (G) SRE3Δ from TP75; and (H) SRE2Δ from TP8. Four set1Δ isolates are shown in each case. (C-H) Data are represented as mean ± SD. Two-tailed paired Student’s t test was used to compare the mean of each sample to cntl, *p < 0.01; **p < 0.005; n.s., not significant.
Fig 6.
Protein interaction network and genetic interactions.
(A) An interaction network of newly identified and previously known mating-type switching factors (Table 1) was obtained from the STRING database (v10.0). The proteins are represented by nodes. Red and blue nodes show factors detected in this screen. White and black nodes show known mating-type switching factors that were not detected in this screen, either because the gene deletions were not in the library, or due to the set thresholds or human error. The line thickness represents the strength of the association (confidence > 0.6). It has been suggested that the presence of the DSB at mat1 is lethal in deletion mutants of rad51, rad52 and rad54 [36, 38]). (B, C) Genetic interactions between swi6 and the identified Class Ib genes. mat1 content was quantified for the indicated double mutants with the h90 (B) or h09 (C) mating-type region. The relative P band intensities (P/(P+M)) were calculated from gels shown in S6 Fig. Red bars represent means ± SD.
Fig 7.
(A) Donor selection controlled by heterochromatin. In the presence of heterochromatin, mat2-P is preferred over mat3-M in M cells due to an increased accessibility of SRE2 to switch-promoting binding factors and to a reduced accessibility of SRE3. In the absence of heterochromatin, SRE3 can stimulate recombination by Swi6-independent binding of Swi2. In P cells, the inhibition to select SRE3 is released. (B) Model of mating-type switching regulated by heterochromatin formation. The histone deacetylases Sir2 and SHREC remove histone acetylation (green). CLRC methylates H3K9 (pink). Swi6 binds to methylated H3K9 and nucleates heterochromatin. CK2 phosphorylates Swi6 (red). Cbp1 binds at specific regions in the mat locus. Clr3 (SHREC) is recruited to phosphorylated Swi6 and Cbp1 binding site. Set1C might affect heterochromatin at SRE3. The Swi2-5 complex binds to Swi6 and Rad51, which regulates mating-type switching directionality. Pof3, Elp6 and Blr2 might also affect switching by histone modification.