(A) FACS-based approach to measure switching rates of HMRα::GFP in sir1∆. Populations of silenced cells were isolated and allowed to divide; as silencing loss occurred, the percentage of expressed cells in the population increased. The distributions of fluorescence intensity per cell at equilibrium are shown in Figure 4—figure supplement 1. (B) For DPB3 MCM2 (blue) (JRY11471), dpb3∆ MCM2 (black) (JRY11550), DPB3 mcm2-3A (green) (JRY11589), and dpb3∆ mcm2-3A (red) (JRY11590), silenced cells were isolated at t = 0 hr, allocated into three separate populations each, and monitored over time. At each time-point, the percentage of expressed cells in each population was determined by flow cytometry (for an example, see Figure 4—figure supplement 2). (C) Silencing-loss rates calculated from (B), as explained in Materials and methods. (D) Silencing-loss rates calculated by monitoring dividing cells with time-lapse microscopy (n > 550 cell divisions per genotype). (E) Similar to (B), except expressed cells were sorted and monitored over time. (F) Silencing-establishment rates calculated from (E), as explained in Materials and methods. (G) Silencing-establishment rates calculated by monitoring dividing cells with time-lapse microscopy (n > 100 cell divisions per genotype). GFP expression levels in expressed cells were calculated by flow cytometry and shown in Figure 4—figure supplement 3. Error bars represent 95% confidence intervals. Two-tailed t-tests were used in statistical analysis of switching rates by sorting, and Yates chi-square tests were used for microscopy (*p<0.05).