Figure 2. Interaction between Brd2 and H2A.Z nucleosomes increases following TSA treatment and is dependent on the bromodomains of Brd2.

A. H2A.Z and H2A nucleosomes were purified as described in Materials and Methods and eluted material was used for Western blot analysis. Prior to harvest cells were treated with TSA or DMSO (vehicle). Brd2 preferentially interacts with H2A.Z nucleosomes and this interaction increases in TSA-treated cells. Brd2 antibody detects both the long and short isoforms (upper and lower bands, respectively) in the INPUT fraction. H3 Westerns show that comparable levels of nucleosomes are loaded between samples and Flag blots show the relative expression/levels of Flag-H2A and Flag-H2A.Z. H4Ac antibody detects the tetra-acetylated form of H4 and is used to show hyperacetylation in TSA-treated samples. B. Mononucleosome IPs were performed using material from cells expressing Flag-H2A.Z and either HA-VPS72 or YFP-Brd2. Anti-HA and -GFP antibodies were used to detect HA-VPS72 and YFP-Brd2 proteins, respectively. Whereas hyperacetylation increases interaction of YFP-Brd2 with H2A.Z nucleosomes, it has no effect on the interaction with HA-VPS72. Total lysates from 293T cells expressing HA-VPS72 and YFP-Brd2 were used to show total expression of the tagged proteins and acetylation status of H4. C. Mononucleosome IPs were prepared from cells expressing Flag-H2A.Z alone (UT) or either wild type (WT) YFP-Brd2 or a mutant version of YFP-Brd2, which contains a single point mutation in each of the bromodomains (BD). Levels of the tagged proteins were detected using both anti-GFP antibody as well as anti-Brd2 antibody (note that the anti-Brd2 antibody detects both the endogenous and exogenous forms of Brd2). Despite the higher relative expression of BD mutant compared to the wild type version, the mutant version does not interact with H2A.Z nucleosomes at detectable levels.