Figure 1. BD2 of BRD4 is required for its interaction with acetylated Twist.

(A) HA-Twist and Flag-BRD4 were co-expressed in HEK293 cells. After treating cells with TSA (2 μM) for 12 hr, Twist, BRD4 and acetylated Twist were immunoprecipitated with HA, Flag and pan-acetylated-lysine (pan-AcK) antibodies, respectively, and analyzed by Western blotting.

(B) HeLa cells stably expressing Flag-Twist were treated with TSA as in (A), Flag-Twist, endogenous BRD4 and acetylated Twist were immunoprecipitated and examined by Western blotting.

(C) Cells were treated as described in (A), endogenous Twist, BRD4 and acetylated Twist were immunoprecipitated and examined by Western blotting.

(D) Schematic depiction of the functional domains of BRD4 and deletion constructs used (top panel). ET stands for extra-terminal domain. Flag-tagged wild-type (WT) or deletion mutants of BRD4 were co-expressed with HA-Twist in HEK293 cells. After immunoprecipitated with HA or Flag antibody, the bound BRD4 or Twist was examined by Western blotting.

(E) Schematic diagram showing the double bromodomain (BD1+BD2) of BRD4 and individual BD constructs used (top panel). Flag-BD1^WT, BD1^YN, BD2^WT and BD2^YN were co-expressed with HA-Twist in HEK293 cells treated with TSA as in (A). Twist and BDs were immunoprecipitated with HA and Flag antibodies, respectively, and analyzed by Western blotting.

(F) HA-Twist and Flag-BRD4 were co-expressed in HEK293 cells treated with TSA as in (A). Twist and BRD4 were immunoprecipitated with HA and Flag antibodies, respectively, in the presence or absence of JQ1 (1 μM) and analyzed by Western blotting.

(G) Cells were treated with TSA as in (A), endogenous Twist and BRD4 were immunoprecipitated with Twist and BRD4 antibodies, respectively, in the presence or absence of JQ1 (1 μM) and examined by Western blotting.

See also Figure S1