Sir3 overexpression leads to hyperclustering of telomeres and is associated with high levels of Sir2, Sir3, and Sir4. (A) Fluorescent images of strains expressing endogenous levels of Sir3 (top) or high levels of Sir3 (bottom). SIR2-GFP (yAT405), GAL1p-SIR3 SIR2-GFP (yAT718), SIR3-GFP (yAT779), GAL1p-SIR3 SIR3-GFP (yAT780), SIR4-GFP (yAT431), and GAL1p-SIR3 SIR4-GFP (yAT720) were grown in galactose synthetic medium before imaging. Immuno-FISH was performed with a Y′-repeat telomeric probe on wild-type (yAT126) and GAL1p-SIR3 (yAT960) strains grown in YPGal. (B) 3D position of telomeres VIL and XIVL relative to each other in living cells expressing endogenous levels of Sir3 (wild type, yAT56) and in strains overexpressing Sir3 (GAL1p-SIR3, yAT690). YFP–tetracycline repressor and CFP–lactose repressor fusions allowed the visualization of tet^op and lac^op arrays inserted at telomeres VIL and XIVL, respectively, as previously described (Bystricky et al., 2005). Cells were grown in galactose before imaging. Shown on the bottom are box plots for distances between telomeres VIL and XIVL. The line in the middle of the box represents the median of the values; the bottom and the top of the box are the 25th and 75th percentiles. The whiskers indicate the minimum and maximum data values. (C) Colocalization of telomeres with Rap1-GFP foci (top): GAL1p-SIR3 RAP1-GFP (yAT208) cells were grown in YPGal for immuno-FISH experiments. Colocalization of Sir3-mCherry foci with Rap1-GFP foci (bottom): GAL1p-SIR3-mCherry RAP1-GFP (yAT330) cells were grown in galactose synthetic medium for live-cell imaging. Bars, 2 µm.