For immunostaining of H3K9me2 and H3K27me3, the cell aggregates were fixed with 4% PFA in PBS for 1 h on ice, washed with PBS, and incubated in PBS containing 0.1% BSA and 0.1% Triton X-100 for 30 min at 4°C for blocking and permeabilization. The cells were incubated with the primary antibody at 4°C for 4 days, and then incubated with the secondary antibody at 4°C for 2 days. For 5 mC immunostaining, the permeabilized cell aggregates were soaked in 4 N HCl, 0.1% Triton X-100 in DDW for 10 min for depurination, washed, and then incubated for blocking. Samples were incubated with the primary and the secondary antibodies at 4°C for 4 and 2 days, respectively. For SYCP3 and SYCP1 immunostaining, the cell spreads were incubated in a blocking solution of PBS containing 5% BSA for 1 h at RT and incubated with the primary antibody overnight at 4°C and then incubated with the secondary antibody and Hoechst for 1 h at RT. For YAP, EpiSCs cultured on glass-bottom dishes coated with FCS were fixed with 4% PFA in PBS for 15 min on ice, washed in PBS, followed by blocking and permeabilization in PBS containing 0.1% BSA and 0.1% Triton X-100 for 15 min at RT. The cells were incubated with the primary antibody at RT for 1 h, and then incubated with the secondary antibody at RT for 30 min. For AP-2γ immunostaining, E7.5 embryos were fixed in 4% PFA in PBS for 3 h on ice, washed in PBS, and then incubated for blocking and permeabilization in PBS containing 0.1% BSA and 0.1% Triton X-100 for 30 min at 4°C. They were incubated with the primary antibody at 4°C for 4 days, and then incubated with the secondary antibody at 4°C for 2 days. The primary antibodies used in this study were: anti-5 mC (1:100; AMM 99021, Aviva), anti-H3K9me2 (1:500; 07-441, Millipore), anti-H3K27me3 (1:500; 07-449, Millipore), anti-BLIMP1 (1:100; sc-47732, Santa Cruz Biotechnology), anti-SYCP3 (1:100; ab97672, Abcam), anti-SYCP1 (1:500; NB300-229, Novus Biologicals), anti-YAP (1:100; 14074, Cell Signaling Technology), anti-E-cadherin (1:100; ab76055, Abcam) and anti-AP-2γ (1:1000; sc-8977, Santa Cruz Biotechnology). The secondary antibodies used in this study were: Alexa Fluor 488 anti-mouse or rat IgG, Alexa Fluor 568 anti-mouse or rabbit IgG, and Alexa Fluor 647 anti-mouse or rabbit IgG (all goat polyclonal; A28175, A11006, A11004, A11011, A21235 and A21245, Invitrogen). The images were captured using a confocal microscope (Zeiss LSM780).

For quantification of immunostaining data, the signal intensity of 5 mC and YAP was quantified using ImageJ software. The signal intensity of 5 mC in the same size area was compared between BLIMP1+ cells and BLIMP1− cells. The signal intensity of YAP in an area of the same size was compared between the cytoplasm and nucleus of each cell. For quantitative analysis, three independent experiments were performed, and in each individual experiment 20 cells were analyzed. P-values were calculated by two-tailed unpaired t-test.