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

Rad22 Coordinates Strand Exchange

<div><p>(A) Effect of the order of Rad22 addition. Various protocols, indicated by numbers, were tested (upper panel). Rad22 was added at the time point indicated by the plus sign (+). The incubation time of each step (arrows) was 5 min. Rad22 was added 2.5 min after the beginning of each indicated step. Samples from each of the reactions (lower panel) were separated by agarose gel electrophoresis. Protocols 1 to 5 gave similarly high levels of strand exchange.</p> <p>(B) Rad22 abrogates the strict requirement for a specific order of protein addition to the strand exchange reaction. The indicated mixtures (1 and 2) were prepared separately, and the reactions were initiated by combining both mixtures and incubating at 37 °C for 120 min.</p> <p>(C) ATP hydrolysis is required for Rhp51-mediated strand exchange. The reactions contained Rad22, Swi5-Sfr1, Rhp51 and RPA, and the indicated nucleotides. The reaction conditions were the same as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060088#pbio-0060088-g001" target="_blank">Figure 1</a>C. The values indicated below each lane in (A) to (C) are average percentages of P and P + JM obtained in three independent experiments. The s.d. is indicated in parentheses.</p> <p>(D) Substoichiometric amounts of Rad22 stimulate Rhp51-mediated DNA strand exchange. An agarose gel containing reactions with increasing concentrations of Rad22 is shown in the upper panel. The graph below shows the yields of the NC product (filled circles) and the total yields of JM intermediates plus the NC product (open circles). The values and error bars are average percentages and s.d. obtained from three independent experiments.</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

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

Rhp51 Per Se Is a Factor That Displaces RPA from ssDNA, and the Two Mediators Synergistically Facilitate This Reaction

<div><p>RPA-saturated ssDNA beads were prepared by incubating immobilized ssDNA with RPA (1 μM) and then removing excess RPA by washing the beads with a buffer. Varying concentrations of each protein (Rad22, Rhp51, and Swi5-Sfr1) or protein mixtures were incubated with RPA-saturated ssDNA in the presence of ATP (2 mM) at 37 °C for 30 min. The css beads were pulled down, and the bound and unbound fractions were analyzed by SDS-PAGE. The fold differences in protein concentrations, indicated above each gel, were based on the concentrations used in the standard strand exchange reaction in this study (i.e., “1-fold” corresponds to 5 μM Rhp51, 0.5 μM Swi5-Sfr1, and 0.5 μM Rad22).</p> <p>(A) Rad22 alone.</p> <p>(B) Swi5-Sfr1 (S/S) alone.</p> <p>(C) Rad22 plus Swi5-Sfr1.</p> <p>(D) Rhp51 alone.</p> <p>(E) Rhp51 plus Rad22.</p> <p>(F) Rhp51 plus Swi5-Sfr1.</p> <p>(G) Rhp51 plus Rad22 plus Swi5-Sfr1.</p> <p>(H) The graph shows the relative amounts of RPA (%) in the unbound fractions. Input RPA (bound + unbound) in the “0-fold sample” was normalized to 100%.</p> <p>(I) The graph shows the amounts of Rhp51 (in micromoles) in the bound fractions. The amount of Rhp51 in the bound fractions was estimated from lanes loaded with 2 μM Rhp51 as a standard. The values and error bars in the graphs are average percentages and s.d., respectively, obtained from three independent experiments. 22, Rad22; 51, Rhp51.</p></div

Mating-type switching related genes.

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

Pull-Down Assays Reveal That the Two Mediators Together Facilitate Rhp51 Presynaptic Filament Formation

<div><p>(A) A titration of RPA to css immobilized on magnetic beads. The indicated amounts of RPA were incubated with css beads (10 μM ssDNA) at 37 °C for 5 min. The protein–DNA complexes were pulled down, and RPA bound to ssDNA was analyzed by SDS-PAGE.</p> <p>(B) Titration of Rhp51 to css beads. The indicated amounts of Rhp51 were incubated with css beads (10 μM ssDNA) at 37 °C for 5 min in the absence or presence of various adenine nucleotides (2 mM). Rhp51-ssDNA complexes were pulled down, and Rhp51 bound to ssDNA was analyzed by SDS-PAGE. The values and error bars in the graphs in (A) and (B) are average percentages and s.d., respectively, obtained from three independent experiments.</p> <p>(C) Adenine nucleotide-dependent ssDNA binding of Rhp51. Rhp51 (5 μM) was pulled down with css beads (10 μM ssDNA) in the absence or presence of various adenine nucleotides (2 mM). The SDS-PAGE image shows bands corresponding to the Rhp51-ssDNA complex in the pull-down fractions.</p> <p>(D–G) RPA has a higher affinity than Rhp51 for ssDNA, and the two mediators synergistically promote Rhp51 presynaptic filament formation in an ATP-dependent manner. Mixtures, indicated below, were prepared and incubated with immobilized ssDNA. Pull-down complexes were analyzed by SDS-PAGE. (D) Rhp51 and RPA. (E) Rhp51, RPA, and Swi5-Sfr1. (F) Rhp51, RPA, and Rad22. (G) Rhp51, RPA, Swi5-Sfr1, and Rad22. The concentrations of proteins used in all assays were 5 μM Rhp51, 1 μM RPA, 0.5 μM Swi5-Sfr1, and 0.5 μM Rad22. A quantitation of bound Rhp51, calculated as a percentage of the amount of Rhp51 in each lane 1 (100%), is shown at right. The values and error bars in the graphs are average percentages and s.d., respectively, obtained from three independent experiments. (C–G) lane 1: input proteins, lane 2: no nucleotide, lane 3: ATP, lane 4: ADP, lane 5: ATPγS, and lane 6: AMP-PNP. Lanes 7, 8, and 9 in (E) and (G) contain Rhp51 (1μM) plus Swi5-Sfr1 (1μM), Rhp51 (1μM) alone, and Swi5-Sfr1 (1μM) alone, respectively, for standards of relative migrations of these proteins.</p></div

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

In Vitro Reconstitution of DNA Strand Exchange

<div><p>(A) Diagram of the DNA strand exchange assay. lss, linear single-stranded DNA.</p> <p>(B) Rad22 can overcome the inhibitory effect of RPA on the Rhp51-Swi5-Sfr1–mediated DNA strand exchange reaction. In lanes 1 to 3, Rhp51 was first incubated with css, and then with Swi5-Sfr1, RPA, and lds. In the lane 1 reaction, SSB was omitted. In lanes 2 and 3, RPA and SSB were added, respectively. In lanes 4 and 5, RPA was first incubated with css and then with Rhp51 and Swi5-Sfr1. Rad22 (lane 5) or mock buffer (lane 4) were added 5 min after incubation with RPA. Lanes 6 and 7 are the same as lanes 4 and 5, respectively, except that SSB was used instead of RPA.</p> <p>(C) Requirements for strand exchange. All protein cofactors are required for the reaction. The protein addition order is indicated above the agarose gel image. When a component was not included, it was replaced by stock buffer. The values indicated below each lane in (B) and (C) are average percentages of NC products (P) and conversions (P + JM) obtained in three independent experiments. Standard deviation (s.d.) is indicated in parentheses.</p> <p>(D) Time course of the strand exchange reaction with various protein components as indicated to the right of each gel image. The reaction procedures including the addition order were the same as in (C) except that the reaction volume was 100 μl. Aliquots (10 μl) were taken at various time points, and the reactions were terminated by adding a stop solution, with a final incubation for 30 min at 37 °C. The graph shows quantifications of P + JM. The values and error bars are the average percentage and s.d. of results from three independent experiments. 22, Rad22; 51, Rhp51; S/S, Swi5-Sfr1.</p></div

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