CAL1-induced ectopic centromeres are epigenetically maintained. (A) IF of lacO cells expressing CAL1–GFP–LacI harvested at day 0 and day 2 after removal of CuSO4 (washout); CAL1–GFP–LacI expression (anti-GFP; green) decreased to undetectable levels at day 2 post-washout. DAPI is in gray. Bar, 5 µm. (B) Experimental strategy used for the experiments in C–E. (C) Metaphase chromosomes from day 0, 2, and 10 show CENP-A at the ectopic lacO sites (white arrow) after loss of CAL1–GFP–LacI. CAL1–GFP–LacI is shown in green, DAPI in blue, and CENP-A in red. (D) Mean ratio of the CENP-A IF intensity in ectopic/endogenous at each time point (n = 10 cells for each day). Error bars represent SEM. (E) Quantification of the mean percentage of cells with visible CENP-A foci at the chromosome arm at each time point. CAL1–GFP–LacI is largely absent from cells on day 2, whereas CENP-A continues to be maintained at the lacO array for several divisions (n = 100, metaphase spread for each time point, error bars show the SD of three independent experiments). (F) Diagram illustrating the strategy to assess self-propagation of ectopic CENP-A chromatin after loss of CAL1–GFP–LacI. (G) Metaphase chromosomes showing the localization of HA–CENP-A (red) and CENP-C (blue) at the ectopic centromere (n = 52 transfected cells, 100% HA–CENP-A recruitment). CAL1–GFP–LacI is shown in green (no signal post-washout), DAPI is in gray. Bars: (C and G) 1 µm.
