New insights into donor directionality of mating-type switching in <i>Schizosaccharomyces pombe</i>

<div><p>Mating-type switching in <i>Schizosaccharomyces pombe</i> entails programmed gene conversion events regulated by DNA replication, heterochromatin, and the HP1-like chromodomain protein Swi6. The whole mechanism remains to be fully understood. Using a gene deletion library, we screened ~ 3400 mutants for defects in the donor selection step where a heterochromatic locus, <i>mat2-P</i> or <i>mat3-M</i>, is chosen to convert the expressed <i>mat1</i> locus. By measuring the biases in <i>mat1</i> content that result from faulty directionality, we identified in total 20 factors required for donor selection. Unexpectedly, these included the histone H3 lysine 4 (H3K4) methyltransferase complex subunits Set1, Swd1, Swd2, Swd3, Spf1 and Ash2, the BRE1-like ubiquitin ligase Brl2 and the Elongator complex subunit Elp6. The mutant defects were investigated in strains with reversed donor loci (<i>mat2-M mat3-P</i>) or when the <i>SRE2</i> and <i>SRE3</i> recombination enhancers, adjacent to the donors, were deleted or transposed. Mutants in Set1C, Brl2 or Elp6 altered balanced donor usage away from <i>mat2</i> and the <i>SRE2</i> enhancer, towards <i>mat3</i> and the <i>SRE3</i> enhancer. The defects in these mutants were qualitatively similar to heterochromatin mutants lacking Swi6, the NAD⁺-dependent histone deacetylase Sir2, or the Clr4, Raf1 or Rik1 subunits of the histone H3 lysine 9 (H3K9) methyltransferase complex, albeit not as extreme. Other mutants showed clonal biases in switching. This was the case for mutants in the NAD⁺-independent deacetylase complex subunits Clr1, Clr2 and Clr3, the casein kinase CK2 subunit Ckb1, the ubiquitin ligase component Pof3, and the CENP-B homologue Cbp1, as well as for double mutants lacking Swi6 and Brl2, Pof3, or Cbp1. Thus, we propose that Set1C cooperates with Swi6 and heterochromatin to direct donor choice to <i>mat2-P</i> in M cells, perhaps by inhibiting the <i>SRE3</i> recombination enhancer, and that in the absence of Swi6 other factors are still capable of imposing biases to donor choice.</p></div

Classification of mutants obtained in the screen according to imprint formation and rearrangements.

<p>Southern blot analysis of <i>Hin</i>dIII-digested DNA. The probe was made from a 10.4 kb <i>mat1 Hin</i>dIII fragment. The positions of <i>Hin</i>dIII sites and primers used for PCR in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s003" target="_blank">S3 Fig</a> (red arrow: <i>mat1</i>-specific; yellow: M-specific and blue: P-specific) are indicated above the blots. Strain PG4045 shows the hybridization pattern of a wild-type <i>h</i>^<i>90</i> 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, <i>swi3Δ</i>. <i>mat3</i>:<i>1</i> (8.2 kb) and <i>mat1</i>:<i>2</i> (6.7 kb) result from the <i>h</i>^<i>+N</i> 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 <i>mat3</i>:<i>1</i> extrachromosomal circular element, or from a duplication of the mating-type region resulting from unequal sister chromatid exchange between <i>mat1</i> and <i>mat3</i>. <i>mat2</i>:<i>1</i> (9.9 kb) might result from a circular minichromosome in the Bioneer <i>swd2Δ</i> mutant. ‘?’ marks a band of unknown origin.</p

Analysis of mating-type switching directionality of Set1C mutants.

<p>(A, B) Spore formation of mutants in each Set1C subunit was assayed by exposure of colonies to iodine vapor. <i>h</i>^<i>90</i> strains are shown in (A) and <i>h</i>^<i>09</i> stains are shown in (B). (C, D, E) Quantification of <i>mat1</i> content estimated by multiplex PCR. The relative P band intensity in each lane (P/(P+M)) was calculated from the data shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s005" target="_blank">S5 Fig</a> for the <i>h</i>^<i>90</i> strains shown in (C); from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s005" target="_blank">S5 Fig</a> for the <i>h</i>^<i>09</i> strains shown in (D) and from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s005" target="_blank">S5 Fig</a> for the strains with mutated <i>SRE</i> elements shown in (E-H). (E) 2×<i>SRE2</i> strains (<i>mat2-P-SRE2 mat3-M-SRE2</i>) were derived from the <i>set1</i>⁺ strain TP126; (F) 2×<i>SRE3</i> strains (<i>mat2-P-SRE3 mat3-M-SRE3</i>) from TP303; (G) <i>SRE3Δ</i> from TP75; and (H) <i>SRE2Δ</i> from TP8. Four <i>set1</i>Δ 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.</p

Screen for mutants defective in donor choice.

<p>(A) Summary of screen. Fluorescence microscopy, multiplex PCR for <i>mat1</i> content and iodine staining were used as described in the text to identify mutants with a <i>mat1</i> content biased towards <i>mat1-P</i> or <i>mat1-M</i>. 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s008" target="_blank">S2</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s010" target="_blank">S4</a> Tables. (B) Examples of cell-type ratios measured by fluorescence microscopy with the dual reporter system using YFP under control of the <i>mfm3</i> M-specific promoter and CFP under control of the <i>map2</i> 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s002" target="_blank">S2 Fig</a> 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 <i>mat1</i> content with a P- and an M-specific primer combined with a <i>mat1</i>-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.</p

Protein interaction network and genetic interactions.

<p>(A) An interaction network of newly identified and previously known mating-type switching factors (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.t001" target="_blank">Table 1</a>) 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 <i>mat1</i> is lethal in deletion mutants of <i>rad51</i>, <i>rad52 and rad54</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.ref036" target="_blank">36</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.ref038" target="_blank">38</a>]). (B, C) Genetic interactions between <i>swi6</i> and the identified Class Ib genes. <i>mat1</i> content was quantified for the indicated double mutants with the <i>h</i>^<i>90</i> (B) or <i>h</i>^<i>09</i> (C) mating-type region. The relative P band intensities (P/(P+M)) were calculated from gels shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s006" target="_blank">S6 Fig</a>. Red bars represent means ± SD.</p

Mating-type switching related genes.

<p>Mating-type switching related genes.</p

Determination of donor-choice preferences in mutants with the <i>h</i>^<i>09</i> mating-type region.

<p>The donor loci in <i>h</i>^<i>09</i> strains are swapped from <i>mat2-P mat3-M</i> to <i>mat2-M mat3-P</i>. Changes in <i>mat1</i> content caused by the Class Ib mutations identified here were estimated by multiplex PCR in the <i>h</i>^<i>09</i> background, from the band intensities shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s004" target="_blank">S4 Fig</a>. 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.</p

Mating-type switching model.

<p>(A) Donor selection controlled by heterochromatin. In the presence of heterochromatin, <i>mat2-P</i> is preferred over <i>mat3-M</i> in M cells due to an increased accessibility of <i>SRE2</i> to switch-promoting binding factors and to a reduced accessibility of <i>SRE3</i>. In the absence of heterochromatin, <i>SRE3</i> can stimulate recombination by Swi6-independent binding of Swi2. In P cells, the inhibition to select <i>SRE3</i> 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 <i>mat</i> locus. Clr3 (SHREC) is recruited to phosphorylated Swi6 and Cbp1 binding site. Set1C might affect heterochromatin at <i>SRE3</i>. 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.</p