A silencing-defective allele of SIR3 is functional for telomere clustering. (A) Mating assays with wild type (WT; yAT7), GAL1p-SIR3 (yAT370), sir3Δ (yAT1196), and GAL1p-Sir3-A2Q (yAT1197). Cells were grown in YPGal plates and replica plated on a lawn of α-mating testers. Telomeric silencing at telVR::ADE2 in wild-type (yAT7), GAL1p-SIR3 (yAT370), GAL1p-Sir3-A2Q (yAT1197), and sir3Δ (yAT1196) strains. Cells were grown in YPGal liquid medium, and dilutions were plated onto YPGal plates. (B) Sir3-A2Q mediates telomere clustering. Fluorescent images of Rap1-GFP in wild-type (yAT7), GAL1p-SIR3 (yAT370), and GAL1p-Sir3-A2Q (yAT1197) cells. Cells were grown in synthetic complete galactose medium overnight, diluted to OD_600nm = 0.2, and imaged at OD_600nm = 1. Numbers correspond to the mean intensity of foci. Quantification of the images was performed using Q-foci. (C) Fluorescent images of an immuno-FISH experiment performed with a Y’-repeat telomeric probe and an anti-GFP on a strain expressing high levels of Sir3-A2Q (yAT1256). (D) ChIP analysis was carried out using an anti-Sir3 to study the spreading of Sir3 on telomere VIR: GAL1p-SIR3 (yAT208), GAL1p-Sir3-A2Q (yAT1205), and sir3Δ (yAT360) strains were grown in YPGal for 48 h. The bar graph represents the enrichment over the mitochondrial locus OLI1 for amplicons at different distances from the TG repeats as indicated. Enrichment at the internal chromosomal locus OGG1 is shown for comparison. The experiment was repeated three times. Error bars represent means ± SEM. Bars, 2 µm.