Cell cross-linking was done by adding 0.8 ml of 37% formaldehyde to 20 ml of overlaying media for 15 min at RT, followed by the addition of glycine to a final concentration of 125 mM (6 (link)). After cross-linking, cells were harvested and then washed twice with 10 ml phosphate-buffered saline (PBS). Cells from one dish were lysed with 1.0 ml IP buffer [150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% NP-40, 50 mM Tris–HCl (pH 7.5) and 0.5 mM DTT] containing the following inhibitors; 10 µg/ml leupeptin, 0.5 mM phenylmethlysulfonyl fluoride (PMSF), 30 mM p-nitrophenyl phosphate, 10 mM NaF, 0.1 mM Na3VO4, 0.1 mM Na2MoO4 and 10 mM β-glycerophosphate. After one wash with 1.0 ml IP buffer the pellet was resuspended in 1 ml IP buffer (containing all inhibitors) and sheared with a sonicator microprobe for 4 rounds of 15, 1 s pulses at output level 7 (Misonix 3000). Sheared chromatin was cleared by centrifugation (10 min at 17 000 g, Eppendorf 5403), split into two 0.5 ml fractions, and was used immediately or stored at −70°C. After adding antibody pre-incubated (30 min at room temp) with or without blocking peptide, 0.5 ml of the sheared chromatin fraction was incubated in an ultrasonic water bath (15 min, 4°C) (Bronson 3510). Tubes were centrifuged (10 min at 17 000 g, Eppendorf 5403) and the supernatant was transferred to fresh tubes containing 20 µl of washed protein A beads (Pharmacia) (19 (link)). The slurry was rotated for 45 min (4°C) and then the beads were washed five times with 1 ml cold IP buffer containing no inhibitors. Yeast chromatin was prepared as previously described (6 (link)).