Reduced Sir4 abundance causes delays in <i>de novo</i> establishment of heterochromatin.

<p><i>(A) De novo</i> establishment was monitored in a single cell establishment assay as described in Osborne <i>et al</i>. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.ref026" target="_blank">26</a>]. Specialized mating strains (JRY8828 and JRY8829) are mated and the behavior of the zygote and its progeny are grown adjacent to a large patch of <i>MAT</i>α cells (ADR22) and their response is monitored in a pedigree assay. JRY8828 has no mating information and will mate as an <i>MATa</i> cell. JRY8829, which has an intact <i>HML</i>α, but is also <i>sir3Δ</i>, will de-repress <i>HML</i>α and mate as an α cell. Immediately after mating the zygote will continue to mate as an <i>MAT</i>α cell because no other mating information is present. The zygote, however, is <i>SIR3/sir3Δ</i> and as soon as <i>HML</i>α is repressed, the zygote and its progeny will behave as a <i>MATa</i> cell and respond to α-factor pheromone which causes cell cycle arrest and the formation of a mating projection. The behavior of the zygote and its progeny are grouped into six pedigree patterns. Pattern 0 denotes a pedigree that silence <i>HML</i>α without dividing, Pattern 1 silence after one division, Pattern 4 after two divisions, and in Patterns 2 and 3 silencing is asymmetric—either the mother or first daughter silence after two divisions. Pattern 5 encompasses all pedigrees that silence after more than two divisions, including those that contain cells that don’t silence within the experiment. <i>(B)</i> Haploid cells that were either <i>SIR4</i> or <i>sir4</i>Δ were mated to form <i>SIR4/SIR4</i> (JRY8828 X JRY8829) and <i>sir4Δ/SIR4</i> (ADR4592 X JRY8829 or JRY8828 X ADR4593, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.s002" target="_blank">S2B Fig</a>) diploid zygotes and were monitored for establishment of silencing at <i>HMLα</i> and categorized as in <i>(A)</i>. The effect of halving the Sir4 levels is significant (p<0.0000001, by the likelihood ratio test). <i>(C) SIR4/SIR4</i> (JRY8828 X JRY8829), <i>sir4Δ/SIR4</i> (ADR4592 X JRY8829), and <i>sir4Δ/sir4Δ</i> (ADR4592 X ADR4593) cells were grown at 25°C, harvested and analyzed by western blot. Two-fold serial dilutions of the <i>SIR4/SIR4</i> sample was analyzed to assess Sir4 concentration. <i>(D)</i> Quantitative mating assays were performed by crossing diploid strains from <i>(B)</i> and a wild type <i>MATa</i> strain (ADR21) to a <i>MATα</i> tester strain (ADR3082). The absolute mating efficiency is the proportion of cells of each query strain that mated and formed colonies on synthetic media lacking amino acids. There is no statistical significance between the mating efficiencies of <i>SIR4/SIR4</i> and <i>sir4Δ/SIR4</i> cells (Student’s two tailed t-test).</p

Increasing Sir4 speeds <i>de novo</i> establishment.

<p>One or two centromeric plasmids, each containing <i>SIR4</i> (pAR646 and pAR722), were transformed into one or both mating strains (JRY8828 and JRY8829) prior to mating. The empty plasmid control represents one empty centromeric plasmid in each strain (pRS313 or pRS316, and see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.s003" target="_blank">S3C Fig</a>). Cells were mated and the resulting zygotes were monitored and categorized as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.g002" target="_blank">Fig 2A</a>. Addition of 2 <i>SIR4-CEN</i> plasmids or 4 <i>SIR4-CEN</i> plasmids were each significantly different from the empty plasmid distribution (p = 0.002 and p = 0.0004, respectively, by the likelihood ratio test). Statistics for every pairwise comparison can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.s008" target="_blank">S1 Table</a>.</p

Mutation of <i>UBP10</i> and <i>YKU70</i> accelerate <i>de novo</i> establishment of heterochromatin.

<p><i>(A) SIR4</i> (JRY8828 and JRY8829), <i>dot1Δ</i> (ADR4631 and ADR4632), <i>ubp10Δ</i> (ADR5087 and ADR5088) or <i>yku70Δ</i> (ADR5841 and ADR5842) cells were mated to produce homozygous zygotes that were monitored and categorized as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.g002" target="_blank">Fig 2A</a>. <i>dot1Δ/dot1Δ</i> distribution is similar to experiments published by Osborne <i>et al</i>. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.ref026" target="_blank">26</a>]. All homozygous deletions are significantly different from the <i>SIR4/SIR4</i> control (p<0.0000001, by the likelihood ratio test). <i>(B)</i> Resultant diploids from <i>(A)</i> were mated to a <i>MATα</i> tester strain (ADR3082) to determine their mating efficiency. The absolute mating efficiency is the proportion of cells of each query strain that mated and formed colonies on synthetic media lacking amino acids. The average and SEM of at least three independent matings are graphed. There is no statistical significance between the mating efficiencies of the four strains (Student’s two tailed t-test). <i>(C)</i> Haploid <i>MATα</i> wild type (ADR22), <i>dot1Δ</i> (ADR6181), <i>ubp10Δ</i> (ADR6182) and <i>yku70Δ</i> (ADR6183) were mated to a <i>MATa</i> tester strain (ADR3081) to determine their mating efficiency. The average and SEM of at least three independent matings are graphed. There is no statistical significance between the mating efficiencies of the four strains (Student’s two tailed t-test).</p

Two models for <i>de novo</i> establishment of heterochromatin.

<p><i>(A) Regulated nucleation</i>. Changes in Sir4 availability and demethylation of histone H3 regulate nucleation of heterochromatin. The abundance and availability of Sir4 is downregulated during pheromone arrest, and telomeres and <i>HML</i>α compete for the available Sir4. When Sir4 is present in extra copies, or is released from telomeres in <i>ubp10Δ</i> or <i>yku70Δ</i> mutants, nucleation occurs faster. If the available Sir4 is reduced in heterozygous <i>SIR4/sir4Δ</i> cells or in <i>rif1Δ rif2Δ</i> cells, that improve telomeric recruitment of Sir4, nucleation slows. These data suggest that recruitment of Sir4 to the <i>HML</i>α silencer is rate limiting for <i>de novo</i> establishment of heterochromatin. Sir4/Sir2 recruitment leads to deacetylation (Ac) of proximal nucleosomes by Sir2, and promotes the recruitment of Sir3 which interacts with the deacetylated N-terminal tails of histone H4. Dot1 adds, and demethylation (DM) removes, methylation (Me) on lysine 79 of histone H3. Demethylation (DM) may occur enzymatically by an unidentified demethylase, by histone exchange, or by deposition of unmethylated histones after DNA replication. Sir3 interacts specifically with unmethylated histone H3, so removal of K79 methylation also promotes Sir3 binding to nucleosomes, and at silencer elements, both deacetylation and demethylation may be required for nucleation of heterochromatin. In <i>dot1Δ</i> mutants, that have no K79 methylation, <i>de novo</i> establishment occurs faster and suggests demethylation of K79 and subsequent recruitment of Sir3 can also be rate limiting for <i>de novo</i> establishment of heterochromatin. Coincident recruitment of Sir3 and Sir4, and their interaction, is required for efficient <i>de novo</i> establishment. <i>(B) Regulated completion of assembly</i>. Changes in Sir4 availability and demethylation of histone H3 regulate transcriptional repression. Kirchamaier and Rine [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.ref020" target="_blank">20</a>] observed a rate limiting step in <i>de novo</i> establishment after spreading of Sir proteins at <i>HMRa</i>, and Katan-Khaykovich and Struhl [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.ref019" target="_blank">19</a>] showed demethylation of K79 on histone H3 is a slow step in <i>de novo</i> establishment. If the abundance of Sir4 acted at a late step in establishment at <i>HMLα</i>, it would suggest that Sir4 occupancy and histone deacetylation may be incomplete in heterochromatin prior to demethylation of histone H3. Recruitment of Sir4 into silent chromatin would allow complete histone deacetylation and be mechanistically linked to histone demethylation and transcriptional repression.</p

<i>DOT1</i> acts upstream or independently of Sir4 abundance.

<p><i>(A) DOT1</i> and <i>SIR4</i> could regulate <i>de novo</i> establishment in one of three ways: Model 1) <i>SIR4</i> inhibits <i>DOT1</i>, and <i>DOT1</i> inhibits <i>de novo</i> establishment, model 2) <i>DOT1</i> inhibits <i>SIR4</i>, and <i>SIR4</i> promotes <i>de novo</i> establishment, and model 3) <i>DOT1</i> and <i>SIR4</i> function in separate pathways to regulate <i>de novo</i> establishment. <i>(B)</i> Cells were mated to create <i>SIR4/SIR4</i> (JRY8828 X JRY8829), <i>sir4Δ/SIR4</i> (ADR4592 X JRY8829), <i>dot1Δ/dot1Δ</i> (ADR4631 X ADR4632), and <i>sir4Δ dot1Δ/SIR4 dot1Δ</i> (ADR4631 X ADR5607 or ADR5640 X ADR4632), and the resulting zygotes were monitored and categorized as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.g002" target="_blank">Fig 2A</a>. The <i>dot1Δ/dot1Δ</i> distribution is significantly different from the <i>sir4Δ dot1Δ</i>/<i>SIR4 dot1</i> and the <i>sir4Δ/SIR4</i> distributions (p<0.0000001 and p<0.0000001, respectively, by the likelihood ratio test). Statistics for every pairwise comparison can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005425#pgen.1005425.s008" target="_blank">S1 Table</a>.</p

Sir4 protein is degraded during a prolonged arrest in G1 and takes two cell cycles to recover after release from the arrest.

<p><i>(A)</i> Asynchronously growing (asyn) wild type (ADR4006) cells were arrested in G1 with 1μg/ml α-factor or arrested in mitosis with 10μg/ml nocodazole at 25°C. Samples were harvested every hour and protein levels were analyzed by western blot. Cdk1 is shown as a loading control. <i>(B)</i> Samples prepared as in <i>(A)</i> were quantified and the average amount of Sir4 (+/- SEM) relative to Cdk1 levels, normalized to the asynchronous (asyn) sample (which was arbitrarily set to 100), of three independent experiments is shown. <i>(C)</i> Asynchronously growing (asyn) wild type (ADR4006) cells were arrested in 1μg/ml α-factor for five hours (αf) and released from the arrest into fresh media at 25°C. Samples were harvested every 30 minutes and protein levels were analyzed by western blot. Cdk1 is shown as a loading control, and the mitotic cyclin, Clb2, is used as a marker of mitosis.</p

Proposed model for redundant inhibition of Esp1 by Pds1 and Slk19.

<p>Our data support a model in which both Pds1 and Slk19 (19) inhibit Esp1. Anaphase onset is triggered by both APC^Cdc20-mediated proteolysis of Pds1 and Cdk1-dependent activation of Esp1. Slk19 and PP2A^Cdc55 inhibit Esp1, and Cdk1 phosphorylation of Esp1 overcomes this inhibition. We speculate that Esp1 phosphorylation triggers the release of Slk19 from the active site of Esp1. Although PP2A^Cdc55 can dephosphorylate Esp1 <i>in vitro</i> and <i>cdc55Δ</i> cells increase Esp1 phosphorylation <i>in vivo</i>, the lack of suppression of <i>cdc55Δ</i> by <i>esp1-3A</i> suggest that PP2A^Cdc55 binding to Separase may be more important for Esp1 inhibition. Slk19 can inhibit Separase either as an uncleaved or cleaved (as depicted) protein.</p

Cdk1^Clb2 phosphorylates Esp1.

<p><b>(A</b>) Esp1 is phosphorylated <i>in vivo</i>. <i>ESP1</i>, <i>ESP1-myc13</i> and <i>pGAL-SWE1 ESP1-13myc</i> cells were grown in YEP + raffinose, arrested in mitosis with nocodazole and switched to YEP + galactose media to induce expression of Swe1. After 1 h, cells were washed in medium lacking phosphate, and grown for 30 min in the presence of [³²P]orthophosphate. Esp1-13myc was immunoprecipitated with 9E10 antibody, run on a polyacrylamide gel and exposed to a phosphorimager screen or immunoblotted. (<b>B</b>) Esp1 contains six minimal Cdk1 consensus sites (S/TP): S13 and T16 (termed <i>N-terminal</i>), T1014, S1027 and T1034 (termed <i>central</i>) and S1280 (termed <i>C-terminal</i>). (<b>C</b>) Mutating Esp1 central residues prevents Esp1 phosphorylation <i>in vivo</i>. Left panels, <i>ESP1</i>, <i>esp1-2A</i>, <i>esp1-3A</i>, <i>esp1-1A</i>, <i>esp1-3A+2A</i>, <i>esp1-2A+1A</i>, <i>esp1-3A+1A</i> and <i>esp1-2A+3A+1A</i> cells were grown in YEP + dextrose, arrested in mitosis with nocodazole and labeled with [³²P]orthophosphate as described in <b>(A)</b>. Right panels, wild-type, <i>cdc55Δ</i> and <i>rts1Δ</i> cells were grown in YEP + dextrose, arrested in G1 with α-factor and released into the cell cycle in the presence of nocodazole. After 90 min cells were labeled with [³²P]orthophosphate as described in <b>(A).</b> Esp1 was immunoprecipitated with anti-Esp1 antibody, run on a polyacrylamide gel and exposed to a phosphorimager screen or immunoblotted. (<b>D</b>) Wild-type and <i>esp1-3A</i> cells were grown to log phase, arrested in G1 with α-factor, and released into the cell cycle (t = 0). α-factor was re-added at t = 60 min to arrest cells in the following G1. Samples were taken for immunoblotting at the indicated timepoints, and run on a polyacrylamide gel containing Phos-tag reagent (top panel), or a standard polyacrylamide gel (bottom panels) and immunoblotted with the indicated antibodies. Note that running and transferring of Phos-tag polyacrylamide gels is inconsistent and cell cycle-dependent changes in protein abundance observed in these panels may not accurately reflect changes in protein abundance. The Esp1 panels from the standard polyacrylamide gel more accurately reflect cell cycle changes in Esp1 abundance. (<b>E</b>) Cdk1^Clb2 phosphorylates the central region of Esp1 <i>in vitro</i>. Esp1 was immunoprecipitated from the strains in <b>(C)</b> growing asynchronously, incubated with γ-[³²P]ATP and purified Cdk1^Clb2-CBP, washed, run on a polyacrylamide gel, and exposed to a phosphorimager screen or immunoblotted with anti-Esp1 antibody. (<b>F</b>) PP2A^Cdc55 dephosphorylates Esp1 <i>in vitro</i>. Esp1 was immunoprecipitated from wild-type cells and phosphorylated with purified Cdk1^Clb2-CBP and γ-[³²P]ATP while immobilized on IgG-coupled magnetic beads. The beads were washed and incubated for the indicated times at room temperature with no addition (yellow lines), TAP-purified PP2A^Cdc55 (blue lines), or PP2A^Cdc55 and okadaic acid (OA) (red lines). The three reactions share a t = 0 sample that was taken before the additions. The dephosphorylation of Esp1 was quantified on a phosphorimager and the extent of dephosphorylation relative to t = 0 (average ± SEM) was graphed. The experiment shown is representative of one of three repeats.</p

Spindle characteristics during mitosis.

<p>Cells with “normal metaphase spindle formation” do not elongate their spindle more than 2 μm in the first 10 minutes after SPB separation and more than 2.5 μm in the first 15 minutes after SPB separation. Cells whose spindles elongate more than 2 and 2.5 μm in these time-intervals display “immediate spindle elongation.” Cells with “failed anaphase” don’t elongate their spindles to 6 μm in the 60 minutes after SPB separation. A small number of cells (15% for cells treated with auxin, and 11% for untreated cells) produce conflicting scores using these two rules (i.e., immediate/normal or normal/immediate in the 10/15 minute intervals), and we categorized these cells manually (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007029#sec018" target="_blank">Materials and Methods</a> for details).</p

<i>ESP1-3D</i> and <i>slk19Δ</i> bypass a Pds1-independent arrest.

<p>The indicated strains were grown to log phase, arrested in G1 with α-factor, and released from the arrest into media containing nocodazole or latrunculin A at 25°C. After 2 h (t = 0), +/- auxin was added to the cultures. Samples were harvested for immunoblotting at the indicated timepoints, run on a polyacrylamide gel, and immunoblotted with the indicated antibodies. <i>cdc55Δ</i> was not analyzed in this experiment because it is defective in both the spindle assembly and morphogenesis checkpoints, and does not arrest in nocodazole or LatA. Pds1-AID migrates adjacent to a background band (indicated by an *).</p