The telomere hypercluster is internal and correlates with more stable silencing. (A) Rap1-GFP hypercluster localization relative to the nuclear pore. Two-color z-stack images were acquired on strains expressing Rap1-GFP, Nup49-mCherry, and either endogenous levels of Sir3 or high levels of Sir3 (yAT222 and yAT223 transformed with the NUP49-mCherry plasmid). The localization of the brightest Rap1-GFP spot in one of the three equal concentric zones was scored on the corresponding focal plane. This experiment was repeated twice: for experiment 1, n_yAT222 = 69 and n_yAT223 = 98, and for experiment 2, n_yAT222 = 77 and n_yAT223 = 173 (n is the number of nuclei analyzed). Error bars represent means ± SEM. Bar, 2 µm. (B) Telomeric silencing at the telVR::ADE2 (YPH499 background) in wild-type (WT; yAT7), GALSp-SIR3 (yAT369), and GAL1p-SIR3 (yAT370) strains. Cells were grown in YPGal medium and plated onto YPGal plates. The color of the colonies is indicative of the state of silencing of the ADE2 reporter gene at telVR: the ADE2 gene is expressed in white colonies and repressed in pink colonies. Fivefold dilution assay (top). Single-colony plating (bottom).