BRD1 forms a HAT complex with HBO1. (A) Purification of the BRD1 complex. Flag-tagged BRD1 protein was partially purified from lysates of K562/Flag-BRD1 cells using an anti-Flag antibody. (B) Western blot analysis of the purified BRD1 complex in panel A by the use of indicated antibodies. (C) Schematic representation of BRD1 and its deletion mutants. Three major domains are indicated. (D) Localization of the binding site in BRD1 for HBO1. 293T cells were transfected with HA-tagged BRD1 mutants with and without Flag-tagged HBO1. Proteins in the lysates of the transfectants were immunoprecipitated with the anti-FLAG antibody and eluted with an excess of Flag peptide. The eluents were analyzed by Western blotting by the use of anti-Flag or HA antibodies. (E) Affinity of BRD1 for the MYST family HATs (HBO1, MOZ, and Tip60) and CBP/p300. 293T cells were transfected with HA-tagged Brd1 together with indicated Flag-tagged HATs. Proteins in the lysates of the transfectants were immunoprecipitated with the anti-FLAG antibody. The immunoprecipitates were analyzed by Western blotting with anti-Flag and HA antibodies. (F) HAT activity of the BRD1 complex. The BRD1 complex was partially purified from lysates of K562/empty vector (Empty) and K562/Flag-BRD1 cells by the use of the anti-Flag antibody and HAT activity on recombinant histones H3 and H4 was evaluated. As a negative control (N.C.), the recombinant histones H3 and H4 were similarly treated without HAT complexes.