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).