Recruitment of CBP to the immobilized chromatin DNA template by GAL4-Eya3 and FLAG-Dach1. (A) Schematic representation of reconstituted chromatin with pIC208-5S/MRG5. rDNA, ribosomal DNA. (B) Reconstituted chromatin (Chr.) or naked DNA (Naked) was end labeled and analyzed by micrococcal nuclease (MNase) digestion. MNase (4 mU for chromatin and 0.8 mU for naked DNA; TaKaRa, Ohtsu, Japan) was added and incubated for the indicated time. The 5S ribosomal DNA repeats were shown by partial EcoRI digestion (lane 7) (12). (C) One-half microgram of chromatin DNA-conjugated Dynabeads was incubated with 20 μg of protein of nuclear extract of NIH 3T3 cells transfected with pfDach1 (lanes 2 and 3) or pCMV-Flag-2 (lanes 5 and 6) in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 1 μg of GAL4-Eya3. Anti-CBP serum, anti-FLAG antibody, and anti-Eya3 serum (20) were used for detection of recruited CBP, FLAG-Dach1, and GAL4-Eya3, respectively.