Competition between Heterochromatic Loci Allows the Abundance of the Silencing Protein, Sir4, to Regulate de novo Assembly of Heterochromatin

Changes in the locations and boundaries of heterochromatin are critical during development, and de novo assembly of silent chromatin in budding yeast is a well-studied model for how new sites of heterochromatin assemble. De novo assembly cannot occur in the G1 phase of the cell cycle and one to two divisions are needed for complete silent chromatin assembly and transcriptional repression. Mutation of DOT1, the histone H3 lysine 79 (K79) methyltransferase, and SET1, the histone H3 lysine 4 (K4) methyltransferase, speed de novo assembly. These observations have led to the model that regulated demethylation of histones may be a mechanism for how cells control the establishment of heterochromatin. We find that the abundance of Sir4, a protein required for the assembly of silent chromatin, decreases dramatically during a G1 arrest and therefore tested if changing the levels of Sir4 would also alter the speed of de novo establishment. Halving the level of Sir4 slows heterochromatin establishment, while increasing Sir4 speeds establishment. yku70Δ and ubp10Δ cells also speed de novo assembly, and like dot1Δ cells have defects in subtelomeric silencing, suggesting that these mutants may indirectly speed de novo establishment by liberating Sir4 from telomeres. Deleting RIF1 and RIF2, which suppresses the subtelomeric silencing defects in these mutants, rescues the advanced de novo establishment in yku70Δ and ubp10Δ cells, but not in dot1Δ cells, suggesting that YKU70 and UBP10 regulate Sir4 availability by modulating subtelomeric silencing, while DOT1 functions directly to regulate establishment. Our data support a model whereby the demethylation of histone H3 K79 and changes in Sir4 abundance and availability define two rate-limiting steps that regulate de novo assembly of heterochromatin

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

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

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

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

<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

A Bayesian reanalysis of the Standard versus Accelerated Initiation of Renal-Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial

Background Timing of initiation of kidney-replacement therapy (KRT) in critically ill patients remains controversial. The Standard versus Accelerated Initiation of Renal-Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial compared two strategies of KRT initiation (accelerated versus standard) in critically ill patients with acute kidney injury and found neutral results for 90-day all-cause mortality. Probabilistic exploration of the trial endpoints may enable greater understanding of the trial findings. We aimed to perform a reanalysis using a Bayesian framework. Methods We performed a secondary analysis of all 2927 patients randomized in multi-national STARRT-AKI trial, performed at 168 centers in 15 countries. The primary endpoint, 90-day all-cause mortality, was evaluated using hierarchical Bayesian logistic regression. A spectrum of priors includes optimistic, neutral, and pessimistic priors, along with priors informed from earlier clinical trials. Secondary endpoints (KRT-free days and hospital-free days) were assessed using zero–one inflated beta regression. Results The posterior probability of benefit comparing an accelerated versus a standard KRT initiation strategy for the primary endpoint suggested no important difference, regardless of the prior used (absolute difference of 0.13% [95% credible interval [CrI] − 3.30%; 3.40%], − 0.39% [95% CrI − 3.46%; 3.00%], and 0.64% [95% CrI − 2.53%; 3.88%] for neutral, optimistic, and pessimistic priors, respectively). There was a very low probability that the effect size was equal or larger than a consensus-defined minimal clinically important difference. Patients allocated to the accelerated strategy had a lower number of KRT-free days (median absolute difference of − 3.55 days [95% CrI − 6.38; − 0.48]), with a probability that the accelerated strategy was associated with more KRT-free days of 0.008. Hospital-free days were similar between strategies, with the accelerated strategy having a median absolute difference of 0.48 more hospital-free days (95% CrI − 1.87; 2.72) compared with the standard strategy and the probability that the accelerated strategy had more hospital-free days was 0.66. Conclusions In a Bayesian reanalysis of the STARRT-AKI trial, we found very low probability that an accelerated strategy has clinically important benefits compared with the standard strategy. Patients receiving the accelerated strategy probably have fewer days alive and KRT-free. These findings do not support the adoption of an accelerated strategy of KRT initiation