Fig. 7. The bromodomain and PWWP domain bind histones. (A) Loading controls of GST-fused recombinant domains of Brpf1 (PHD finger, bromodomain, or PWWP domain) used in histone-binding assays (B-D). Relevant bands are indicated with asterisks. (B) Coomassie-staining of histones retained on glutathion beads without (A) or with (B) indicated GST-Brpf1 domains. Left lane shows 10% input of core histones used per assay. (C) Binding of purified H2A or H2B from calf serum with indicated GST-Brpf1 domains, analyzed by Coomassie staining. (D) Binding of core histones from untreated or butyrate-treated HeLa cells with indicated GST-Brpf1 domains, analyzed by western blotting with anti-H2AK5Ac (upper panel) or anti-H2A (lower panel) antibodies. The PWWP domain binds regular and hyperacetylated H2A equally well (compare lanes 5 and 6 of lower panel), whereas the bromodomain preferentially binds hyperacetylated H2A (compare lanes 3 and 4 of lower panel). This is also reflected in the higher relative signal intensity obtained with the anti-H2AK5Ac and the anti-pan H2A antibodies (compare upper and lower bands of lane 4 with those of lanes 6 and 8).