A truncated Sir4C is sufficient for silencing at <i>HML</i> and <i>HMR</i>.

<p>A) Scheme of Sir4 indicating important domains and their interactions. The N-terminal domain of Sir4 (Sir4N) is in red, the C-terminal domains in green (full Sir4C = 747–1358; light green PAD = 950–1262; dark green coiled coil domain = 1262–1358). B) Scheme of plasmids expressing Sir4 constructs. The plasmid's original promoter and terminator were replaced with a 1 kb sequence of the <i>SIR4</i> 5′ region and 250 bp of the 3′ region containing the endogenous promoter/terminator information. The same plasmid construct with different markers was used as needed. C) Silencing at <i>HML</i> of strains with various Sir4 domains was assayed by quantitative mating to a tester strain (GA858). The endogenous <i>SIR4</i> copy was full length (<i>SIR4</i>; GA503), a C-terminal deletion (<i>sir4N</i>; GA5809) or a complete deletion of Sir4 (<i>sir4</i>Δ; GA5822). C-terminal or full length Sir4 was added back on a plasmid. Mating efficiency was normalized to the wild-type strain; data represent mean value ± s.e.m, n.d. undetermined values. D) Plasmids similar to (C), but silencing at <i>HMR</i> was assayed using a <i>TRP1</i> reporter (GA484, GA6072, GA5886). Serial dilutions of transformed strains were grown on control plates selecting for the plasmid only or on plates selecting for the plasmid and growth without tryptophan (monitoring repression of <i>TRP1</i>).</p

Summary of silencing phenotypes.

<p>Summary of silencing phenotypes of Sir4 C-terminal constructs on different reporters and backgrounds. Qualitative silencing strength is given (+++: same level as full length <i>SIR4</i>, ++intermediate silencing, +weak silencing, −no silencing (same as <i>sir4</i>Δ), n.d. undetermined value).</p

Sir4N is the major site of phosphorylation.

<p>A) Scheme of Sir4 as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002727#pgen-1002727-g001" target="_blank">Figure 1A</a>, indicating identified phosphoacceptor sites of full-length Sir4. Serine 63 and 84 mutated in subsequent experiments are indicated in red. Sites having the [S/T*]-P consensus are in bold. B) Relative quantification of the enrichment of Sir4 phosphopeptides in G2/M over cycling cells by LC-MS of a trypsin and of a combined AspN/chymotrypsin digest of a Sir4-IP experiment. For each digest the extracted ion chromatograms were integrated and the ratios of peptides detected in G2/M versus log growing cells were calculated for five phosphorylated as well as five non-phosphorylated Sir4 peptides. The ratio average of the five non-phosphorylated peptides of each digest was expected to be 1 and the corresponding correction factors were used for normalization of the phosphorylated peptides. For the five non-phosphorylated peptides the ratio average is displayed with error bars as standard deviation. C) <i>In vitro</i> phosphorylation followed by partial trypsin digestion of recombinant Sir4N and indicated Sir4N phosphosite mutants. The tryptic peptides were separated by high resolution SDS-PAGE and analyzed by radiography. Peptide sequences to the right indicate the migration pattern of trypsin-digested <i>in vitro</i> phosphorylated standard peptides containing the indicated phospho-serine or -threonine residues D) Interaction of Sir4N with known interaction partners was analyzed by yeast two-hybrid analysis. Sir4N and Sir4N^GG or Sir4N^DD mutants were used as prey, for Yku80, Sir1 and Sif2 bait constructs that induce expression of the β-galactosidase gene upon interaction. At least three independent experiments were averaged for each value; data represent mean value ± s.e.m. E) Sir4N fragment indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002727#pgen-1002727-g007" target="_blank">Figure 7A</a> and the respective mutants were expressed and purified from <i>E. coli</i>. DNA binding was performed and analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002727#pgen-1002727-g005" target="_blank">Figure 5A</a>; data represent mean value ± s.e.m of three independent experiments. F) Sir4N fragments were bound to 6 mer arrays of nucleosomes and challenged with increasing amounts of MNase as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002727#pgen-1002727-g005" target="_blank">Figure 5D</a>. Quantification of two independent experiments was used, data represent mean values.</p

Sir4N phosphoacceptor site mutants show increased accumulation of active states and overall derepression of TPE.

<p>A, B) Single colony streaks for <i>ADE2</i> color assay monitor silencing of the indicated mutant strains (GA6018, GA5887, GA5888, GA5822, GA503). C,D) Quantification of cells swapping from a silent red to a de-repressed white state or vice versa. Single colonies were grown for the indicated time and dilutions plated on YPAD to monitor colony color. E) Single white or red colonies as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002727#pgen-1002727-g008" target="_blank">Figure 8C/8D</a> were grown overnight and then spotted in dilution series onto YPAD plates for <i>ADE2</i> color development, or on plates lacking uracil. To test rapamycin sensitivity, cells were additionally spotted onto SC plates containing 2.5 nM rapamycin. Colony growth was quantified as % of survivors growing on plates lacking uracil or containing 2.5 nM rapamycin. Results are plotted in the bar graph (two independent isogenic strains, each scored in 4–8 experiments; combined data are represented as mean value ± s.e.m). We note that the <i>sir4^P2A</i> effect is stronger at Tel5R than at Tel7L, possible because the silencing of reporters at Tel5R is much weaker to begin with <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002727#pgen.1002727-Gottschling2" target="_blank">[99]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002727#pgen.1002727-Mondoux1" target="_blank">[100]</a>. F) Relative quantification of mRNA of <i>HML-APLHA1</i> and the three indicated subtelomeric genes (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002727#pgen-1002727-g003" target="_blank">Figure 3C</a>) in white or red colonies recovered from <i>SIR4</i>, <i>sir4</i>Δ and <i>sir4^P2ADD</i> red and white colonies (GA503, GA5822, GA5887). Data from three biological replicates are represented as mean values ± sem G) Microarray analysis of <i>SIR4</i>, <i>sir4</i>Δ and <i>sir4^P2ADD</i> red and white colonies (GA503, GA5822, GA5887). Plotted are the zero centered fold changes of log2 expression values of genes as a function of their distance from the telomere in relation to data for an isogenic <i>SIR4⁺</i> strain. Black spots represent single genes, the red line is lowess smoothed over all genes.</p

Sir4C has reduced affinity for DNA and chromatin and protects linker DNA less from MNase attack.

<p>A) Increasing amounts of Sir2–Sir4 or Sir2–Sir4C complexes were titrated into a fixed amount of 167 bp 601-Widom Cy5-labeled DNA. Samples were separated by native agarose gel electrophoresis and visualized by Cy5 fluorescence. Binding in three independent experiments was quantified by measuring the disappearance of the unbound DNA and normalized to input; data represent mean value ± s.e.m. B) Sir2–Sir4 and Sir2–Sir4C complexes were titrated into constant amounts of 6 mer arrays of unmodified nucleosomes. Samples were analyzed as (A); chromatin was visualized by SybrSafe staining. C) Sir2–Sir3–Sir4 and Sir2–Sir3–Sir4C complexes were titrated into constant amounts of 6 mer arrays of unmodified nucleosomes as in (B), with only one experiment analyzed. D) Indicated concentrations of Sir2–Sir4 or Sir2–Sir4C were bound to chromatin as in (B) and incubated with increasing amounts of MNase for 10 min on ice prior to deproteinization. DNA was analyzed by agarose gel electrophoresis and SybrSafe staining. The amount of full length 6 mer DNA was quantified to monitor degree of digestion. Data from at least three experiments are represented as mean value ± s.e.m.</p

Sir4C supports Sir3 focus formation <i>in vivo</i>.

<p>A, B) Sir3-EGFP foci were monitored in logarithmically growing cultures using live microscopy. Sir3-EGFP was tagged at its endogenous locus and the strains carried the indicated forms of <i>SIR4</i> (GA3128, GA6287, GA6288). Full-length Sir4 or Sir4C were added back on plasmids. Images were quantified by counting cells having >3 Sir3 foci at a low signal threshold or >1 Sir3 focus at a high threshold of equally treated images (n>240 cells/sample; >2 independent experiments; data represent mean value ± s.e.m). C) Single focal planes of deconvolved images of Sir3-EGFP as above; size bar 1 µm.</p

Sir4C is not sufficient for silencing at compromised <i>HM</i> loci.

<p>A) Quantitative mating assays were performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002727#pgen-1002727-g001" target="_blank">Figure 1C</a> with strains additionally carrying full deletions of <i>YKU70</i> or <i>SIR1</i> (GA6069, GA6070, GA6071, GA6062, GA6063, GA6064). Mating was normalized to wild-type cells and at least three independent experiments were quantified; data represent mean value ± s.e.m. # indicates values below 10⁻³, n.d. undetermined values. B, C, D) Testing silencing of compromised <i>HMR</i>: full <i>sir4</i> deletion or endogenous <i>sir4N</i> were complemented with <i>SIR4</i>, <i>SIR4C</i> or a <i>SIR4N-C</i> fusion in strains carrying a <i>TRP1</i> reporter at <i>HMR</i>. The <i>HMR-E</i> silencer carried a deletion of either the B (Abf1 binding; GA485, GA6888, GA6899) or A (Orc1-Sir1 binding; GA486, GA6890, GA6891) site. Dilution series for repression were performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002727#pgen-1002727-g001" target="_blank">Figure 1D</a>.</p

Sir4C is not sufficient for silencing at telomeres.

<p>A) Telomeric silencing was monitored by a Tel7L::<i>URA3</i> reporter gene (GA503, GA5809, GA5822) expressing the indicated proteins from pRS, including the SIR4N-C fusion. Growth on plates containing 5 nM rapamycin (rapa) was also monitored. B) Relative mRNA levels of three different subtelomeric genes and <i>HML-ALPHA1</i> were measured using QPCR. Bars represent averages of biological triplicates, data represent mean value ± s.e.m. C) Scheme of the <i>HM</i> loci and telomeres analyzed, indicating additional recruiting elements and distances of promoters from nucleating elements.</p

Sir4C can form a stable and active SIR complex in a recombinant system.

<p>A) SIR complexes as indicated were purified form co-infected insect cells. 1 µg of each complex was run on a SDS-PAGE and visualized by Coomassie staining. B) Purified Sir2–Sir3–Sir4 and Sir2–Sir3–Sir4C complex were incubated with histone octamers acetylated at H4K16 with or without the essential cofactor NAD. The deacetylation reaction was stopped after various time points by the addition of sample buffer and monitored by immuno blotting for H4K16^ac and H3, for equal loading. C, D) Sir2–Sir3–Sir4 or Sir2–Sir3–Sir4C complexes were analyzed by density gradient sedimentation. Fractions were run on 4–12% NuPAGEs Novex Bis-Tris Gels and stained with Sypro Ruby dye. Intensities of Sir2, Sir3 and Sir4 full length proteins were quantified (QuantityONE) and plotted in line graphs. The asterisk in D) indicates a Sir4 degradation band that runs very closely to Sir3.</p