Western blot analysis

Cells were grown at 30°C in 2 mL of SC–Ura medium for overnight and then measured the optical density at 600 nm (OD₆₀₀) and inoculated into fresh medium at initial OD₆₀₀ of 0.5 in 3 mL. After 4 h, 1 OD₆₀₀ units were harvested at log-phase when OD₆₀₀ was around 1. The cells were treated with 1 mL of 0.2 N NaOH for 5 min at RT and centrifuged at 15,000 rpm for 1 min. The pelleted cells were suspended in 50 μL of 1× NuPAGE LDS Sample Buffer (Invitrogen) containing 100 mM DTT and heated at 100°C for 5 min. For the analysis of the TAP-tagged proteins, two-fold serially diluted lysates were analyzed as described below, and we confirmed that 0.025 OD₆₀₀ units provide appropriate signal in the linear range. The extract diluted 8-fold with 1× NuPAGE LDS Sample Buffer, corresponding to 0.025 OD₆₀₀ units, was separated by polyacrylamide gel electrophoresis with lithium dodecyl sulfate (SDS-PAGE) on NuPAGE 4%–12% Bis-Tris Gel (Invitrogen). For the analysis of the GFP-tagged proteins, 0.2 OD₆₀₀ units were subjected to SDS-PAGE. The separated proteins were transferred onto PVDF membrane using the iBlot Transfer Stack PVDF membrane (Invitrogen). The blotted membrane was treated with PBST [1× PBS, 0.1% Tween 20] for 10 min, and then blocked with 4% skim milk in PBST for 1 h at RT. The TAP-tagged proteins were probed with PAP (Sigma-Aldrich) (1:2,000) for 1 h at RT. The GFP-tagged proteins were probed with anti-GFP antibody (Roche) (1:1,000) and peroxidase-conjugated secondary antibody (Nichirei Biosciences) (1:1,000) for 1 h at RT. After washing the membrane with PBST for 5 min for three times, chemiluminescence was induced by adding 500 μL of SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) on the membrane and detected using LAS-4000 image analyzer (Fujifilm) and ImageQuant LAS 4000 (GE Healthcare). Quantification of the band intensity was carried out using ImageQuant TL (GE Healthcare) and the fold change was calculated after background subtraction. After washing the membrane with sterile water for 5 min for three times, total proteins were visualized by Coomassie staining with SimplyBlue SafeStain (Invitrogen) to confirm equal loading of proteins. The stained membrane was digitized using LAS-4000 image analyzer and ImageQuant LAS 4000. Representative blots and corresponding Coomassie stains are shown in S11 Fig.

For the analysis of histone H2A/H4 and the Bet2–Bet4 heterodimer, the harvested cells were treated with 1 mL of 0.2 N NaOH for 5 min at RT. Cells were suspended in 1× NuPAGE LDS Sample Buffer (Invitrogen) and then heated at 70°C for 10 min. The supernatant corresponding to 0.5 OD₆₀₀ units was labeled with EzLabel FluoroNeo (ATTO) and subjected to SDS-PAGE, followed by Western blot with PAP (Sigma-Aldrich) (1:2,000) as described above.