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1.
FIG. 7.

FIG. 7. From: Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2 .

G1/S-phase cells mutated for SLX5 have a reduced ability to form colonies. Individual cells were taken from a liquid culture growing logarithmically and positioned on rich medium by micromanipulation. Resulting colony growth from these founder cells was monitored microscopically after incubation at 30°C. Error bars are based on two or three replicates from independent cultures.

Russell P. Darst, et al. Mol Cell Biol. 2008 February;28(4):1361-1372.
2.
FIG. 3.

FIG. 3. From: Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2 .

Epitope-tagged Slx5 protein resided in the nucleus in structures distinct from the telomeres or nucleolus. Strain LPY8908 contains plasmid pLP1737 with the SLX5-V5 construct on a GAL1 promoter (45). Cells were grown in 2% raffinose to late log phase. To induce, galactose was added to 2% and cultures were incubated at 30°C for the indicated times. Indirect immunofluorescence was performed as previously described (14), with antiserum to Sir2 (fluorescein isothiocyanate-conjugated secondary antibody, in green) and antiserum to the V5 epitope (Texas Red-conjugated secondary antibody, in red). DNA is stained with DAPI (blue).

Russell P. Darst, et al. Mol Cell Biol. 2008 February;28(4):1361-1372.
3.
FIG. 2.

FIG. 2. From: Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2 .

Evaluating Sir2 and Slx5 interaction in vitro. (A) GST and GST-Sir2 were purified from bacteria with GST-Sepharose. Purified proteins were incubated with whole-cell extracts from yeast with or without Slx8 or chromosomally tagged HA-Slx5. Bound protein was subjected to SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of HA-Slx5 was performed with anti-HA monoclonal antibody. Note the enhanced interaction in the slx8Δ mutant, perhaps reflecting decreased competition for binding. (B) The same experiment was performed in the absence of Sir3 and Sir4, Slx8, or Sir2. As before, deletion of SLX8 enhances the interaction, even in the absence of Sir3 and Sir4. Although the interaction appeared to be enhanced in the absence of Sir2, more Slx5 was apparent in the input as well. Immunoblot analysis of GST was performed with anti-GST polyclonal antibody.

Russell P. Darst, et al. Mol Cell Biol. 2008 February;28(4):1361-1372.
4.
FIG. 6.

FIG. 6. From: Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2 .

Restoring silencing in slx5Δ mutant strains. (A) Although deletion of SLX5 caused expression of the telomeric URA3 reporter and toxicity on 5FOA-containing medium, this phenotype was fully suppressed by SUM1-1. wt, wild type. (B) Deletion of RPD3 improves telomeric silencing, as previously reported (63), and bypasses the requirement for SLX5. Single mutants and two independently derived rpd3Δ slx5Δ double mutants are shown. (C) Tethering Sir2 near the telomere restores silencing to slx5Δ mutants. The GBD-Sir2 fusion binds the UASg sequence placed downstream of the URA3 reporter as diagrammed above. Presence of the GBD-Sir2 fusion construct restored silencing (growth on 5FOA) in the slx5Δ mutant, even by Sir2 mutant proteins with reduced catalytic activity (17).

Russell P. Darst, et al. Mol Cell Biol. 2008 February;28(4):1361-1372.
5.
FIG. 1.

FIG. 1. From: Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2 .

GAD-Slx5 interaction with GBD-Sir2. Sir2 is diagrammed above. The core contains residues 244 to 457 of Sir2. GBD-Sir2 contains residues 74 to 562. All sir2 point mutants were tested in this construct. GBD fusions were expressed from a TRP1-marked plasmid. GAD-Slx5 fusion was expressed from a LEU2-marked plasmid. The growth plate lacks leucine and tryptophan. The interaction plate additionally lacks adenine and histidine to assay simultaneously for activation of the reporter genes GAL1-HIS3 and GAL2-ADE2, which share minimal promoter sequence similarity yet are highly induced by the same activator, Gal4, thus eliminating promoter-specific false positives (29). Growth on this plate indicates a physical association between the GBD-Sir2 fusion and GAD-Slx5. Point mutants demonstrate that the interaction is not solely dependent on catalytic activity but is dependent on at least one residue outside the Sir2 catalytic domain.

Russell P. Darst, et al. Mol Cell Biol. 2008 February;28(4):1361-1372.
6.
FIG. 8.

FIG. 8. From: Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2 .

Silencing defect of slx5Δ mutants is linked to the SUMO pathway. (A) The 2μm plasmid is not required for the silencing defect of the slx5Δ mutant. A 5FOA silencing assay was performed as described in the legend to Fig. 4. As shown in the left part of the panel, curing slx5Δ mutants of the 2μm plasmid (cir0) rescued clonal lethality; however, the right part of the panel indicates that silencing was not restored. (B) The slx8Δ mutant has a moderate silencing defect. The slx5Δ slx8Δ double mutant also has a silencing defect, indicating that loss of silencing is not due to unregulated activity of one complex member in the absence of the other. (C) Silencing of a telomeric URA3 reporter is lost in ulp2Δ mutants. (D) Mutants of SIR2 require ULP2 for viability. Haploid products of a heterozygous diploid (SIR2/sir2Δ::TRP1 ULP2/ulp2Δ::HIS3 + URA3 SIR2 plasmid) with the indicated genotypes and the URA3 SIR2 plasmid pLP37 were grown in medium lacking uracil (+ pSIR2) to maintain the plasmid or in 5FOA (no plasmid) to select against the plasmid. Little to no viability was observed for multiple independent double mutants.

Russell P. Darst, et al. Mol Cell Biol. 2008 February;28(4):1361-1372.
7.
FIG. 4.

FIG. 4. From: Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2 .

Slx5 contributes to transcriptional silencing. (A) Deletion of SLX5 disrupted telomeric silencing. URA3 gene expression is toxic on 5FOA. Wild-type (wt) yeast silenced the telomere VR-proximal URA3 reporter and grew on 5FOA (silencing), whereas silencing mutants such as the sir2Δ mutant did not. The slx5Δ mutant grew on 5FOA when it lacked the URA3 gene (top) but not in the presence of the telomeric URA3 reporter. (B) Red indicates silencing at the telomeric VR-proximal ADE2 reporter gene. Wild-type cells formed both red and white colonies; both the sir3Δ and slx5Δ mutants formed uniformly white colonies. Note the colony size heterogeneity of slx5Δ cells, a characteristic of the mutants that has been described previously (45). (C) Deletion of SLX5 disrupted rDNA silencing. Growth on medium lacking uracil (silencing) indicates the expression of an mURA3 reporter located in nontranscribed spacer 1 (NTS1) of one unit of the rDNA array. Two independent isolates each of the sir2Δ mutant, the wild type, and the slx5Δ mutant are shown. (D) Deletion of SLX5 did not disrupt mating-type silencing even in the presence of the hmrΔE mutation, which weakens silencing at the HMR locus. Growth on medium lacking tryptophan (silencing) indicates loss of silencing of a TRP1 reporter integrated at HMR, as in the sir1Δ and sir2Δ mutants but not the slx5Δ mutant. (E) Quantification of mRNA by reverse transcription and real-time molecular amplification. Experiments were performed with strains with a telomeric URA3 reporter gene and a chromosomal deletion of the ura3-1 locus. Bars represent the URA3 or YFR057W cDNA signal minus control reaction mixtures not containing reverse transcriptase, divided by ACT1 cDNA. No enrichment of URA3 signal was detected in the wild type. For the primer sequences, see Table S3 in the supplemental material. Error bars are standard deviations from two independent experiments.

Russell P. Darst, et al. Mol Cell Biol. 2008 February;28(4):1361-1372.
8.
FIG. 5.

FIG. 5. From: Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2 .

Molecular hallmarks of telomeric silencing are largely intact in slx5Δ mutants. (A) Sir2 was expressed at the wild-type (wt) level in slx5Δ cells. The top part of the panel is an anti-Sir2 protein immunoblot assay showing Sir2 in lysates produced from four, two, or one relative amount of cells; the bottom part of the panel is Ponceau stain as a loading control. (B) Silencing protein occupancy at telomere VIR was evaluated by ChIP with antiserum to Sir2. Sir2-immunoprecipitated (IP) DNA was quantified by real-time molecular amplification. Error bars equal 1 standard deviation of three to five independent experiments at each locus. (C) ChIP with antiserum to Sir3. Error bars, where shown, equal 1 standard deviation from two independent experiments. (D) Histone modification state was evaluated by ChIP with antiserum to acetylated histone H4 lysine 16. Error bars equal 1 standard deviation from two independent experiments. The primers for 0.2 kb from telomere VIR were not included because the AcH4K16 immunoprecipitation was less efficient at that locus in all of the strains tested, suggesting altered nucleosome occupancy at that position. (E) Sir3GFP fusion protein, expressed from an in-frame integration of the GFP coding sequence into the chromosomal SIR3 locus (27), was visualized in live cells. Three-dimensional deconvolution was used to resolve telomeric Sir3GFP foci. Each image is a collage of four representative z sections superimposed on their corresponding differential interference contrast images. In addition to the apparently normal cells shown here, the slx5Δ population included many dead cells and misshapen cells that did not have clear Sir3GFP foci.

Russell P. Darst, et al. Mol Cell Biol. 2008 February;28(4):1361-1372.

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