SUMOylation of Sir4 supports its incorporation into subtelomeric chromatin during mitosis. (A–C) His₈-SUMO–conjugates were affinity purified from the indicated strains containing Sir4-V5₃ (A and B) or the sir4^K1037R-V5₃ SUMO site mutant (C). Levels of Sir4 and sir4^K1037R in the cell lysates (L) and Sir4-V5₃-SUMO and sir4^K1037R-V5₃-SUMO in eluates (E) were examined by anti-V5 Western blotting. (D) Tethering of Tel14L to the NE in the indicated strains was assessed as described in Fig. 6. WT data are the same as that used in Fig. 6 A. Graphs represent at least three biological replicates where n = 50 cells/replicate/cell cycle stage. Error bars represent SD. (E) ChIP analysis was performed using synchronized cultures of the indicated strains at various cell cycle stages, including G1-phase (α-factor arrested cells), S-phase (30 min after α-factor release), and M-phase (60 min after α-factor release). Analysis was performed on a region 0.5 kb from Tel6R. Graphs represent at least three biological replicates. Error bars represent SEM. Asterisks: significant change relative to WT using a two-tailed Student’s t test. *, P ≤ 0.05; ***, P ≤ 0.001. Ana/Telo, Anaphase/Telophase; Phos, phosphorylation.