Fig. 7.

19X disrupts the interaction between Brd3 and acetylated GATA1, blocks erythroid maturation, and impairs GATA1 binding to chromatin. (A) GST-BD1 was exposed to nuclear extracts from 293T cells expressing GATA1 and CBP in the presence of the indicated concentrations of 19X. Bound material was analyzed by anti-GATA1 Western blot. Input equals 2.5%. (B) GATA1 peptides were incubated with nuclear extracts from 293T cells expressing full-length HA-Brd3 in the presence of the indicated concentrations of 19X. Retained material was probed by anti-HA Western blotting. Input equals 2.5%. (C) GATA1 peptide binding assays with GST-BD1 in the presence of varying concentrations of 19X. Bound material was analyzed by Western blotting using GST antibodies. Input equals 4%. (D) Anti-HA immunoprecipitation of nuclear extracts from 293T cells expressing GATA1 and HA-Brd3 in the presence of 19X, followed by Western blotting with anti-GATA1 or anti-HA antibodies. Input equals 2.5%. (E) qRT-PCR measuring fold changes in mRNA levels of GATA1 target genes Hbb-b1, Eraf, Slc4a1, and Zfpm1 (Right) and control genes Pabpc1 and Brd3 (Left) in G1E and estradiol-treated G1E-ER4 cells. Prior to harvesting, cells were grown in the presence of the indicated concentrations of 19X for 24 h. The data shown are the average of two to four independent experiments. (F) ChIP-qPCR analysis of G1E and estradiol-treated G1E-ER4 grown in the presence or absence of 1 μM 19X for 24 h. The data shown are the average of three to four independent experiments. Asterisks indicate p < 0.05. ns, not significant. All error bars denote standard deviation.