Figure 5. BRD4 and GATA4 interact in a bromodomain-independent manner to control the master regulator of mitochondrial homeostasis Ppargc1a.

(A) Gene reporter assay showing activation of indicated luciferase reporters upon addition of plasmids encoding indicated proteins. Statistical significance is shown between control and GATA4 + BRD4 vs. all other individual conditions (n=4). (B) Relative expression by RT-qPCR in neonatal rat ventricular myocytes. Statistical significance is indicated between siControl and siGata4 + siBrd4 vs. all other individual conditions (n=3). One-way ANOVA analysis coupled with a Tukey test was used to assess significance. (C) Immunoprecipitation (IP) of endogenous protein from human cardiac progenitor cells using anti-GATA4 or anti-IgG antibody and immunoblotting (IB) with anti-BRD4 or anti-GATA4 antibody demonstrates endogenous BRD4 co-immunoprecipitates with GATA4 but not IgG. (D) Immunoprecipitation of FLAG-BRD4 overexpressed in HEK293 cells followed by immunoblotting with anti-GFP or anti-FLAG antibodies demonstrates GFP-GATA4 still co-IPs with BRD4 even in the presence of increasing doses of JQ1 or DMSO as control. (E) Immunoprecipitation of FLAG-BRD4, FLAG-BRD4^N140A/N433A, and FLAG-BRD4^Y97A/Y390A in HEK293 cells followed by immunoblotting with anti-GFP or anti-FLAG antibodies indicates co-immunoprecipitation of BRD4 mutants with GFP-GATA4.

For A and B, * represents p<0.05, ** represents p<0.01, *** represents p<0.001 and **** represents p<0.0001 for indicated comparison.