FIG. 5.
ERRα/Bcl3/PGC-1α form a trimeric complex on an ERRα-responsive element in cardiac myocytes. Coimmunoprecipitation experiments were performed by cotransfecting combinations of Flag-ERRα, PGC-1α-myc, and Bcl3 in COS7 cells as indicated. Antibodies against the Flag epitope (A and B) and myc-epitope (C) were used for coimmunoprecipitation. The extracts (Input) from COS7 cells and the proteins from the immunoprecipitations (IP) were analyzed by immunoblotting (IB) as noted. Each coimmunoprecipitation was repeated three times, and a representative result is shown. (D) A schematic representation of the rat PDK4 promoter region is shown at the top of the panel with arrows representing primers used for ChIP studies. PDK4P represents the region containing the ERRα responsive element, and the intergenic control (IC) represents an upstream region that does not contain any putative ERRα-responsive consensus element (negative control). The numbers indicate the corresponding nucleotides. The middle panel shows a ChIP assay performed on chromatin extracted from rat neonatal cardiac myocytes using IgG or antibodies against ERRα, PGC-1α, and Bcl3 as labeled. Reimmunoprecipitation (Re-IP) was performed on chromatin immunoprecipitated by antibody against ERRα and subsequently reimmunoprecipitated using IgG or antibodies against PGC-1α and Bcl3. Crude chromatin (Input) and immunoprecipitated chromatin were analyzed by PCR with primers (arrows) for IC, PDK4P, and rat 36B4 genome. Quantification using SYBR green real-time PCR is shown in the bottom panel. The relative increase in enrichment represents fluorescent signal generated by PDK4P (calculated as a percentage of input) divided by fluorescent signal generated by IC (calculated as a percentage of input and set to 1.0) for each antibody. Bars represent the mean relative increase in enrichment (± SEM) from three independent experiments. *, significant differences between IC and PDK4P values for each antibody. (E) ChIP was performed on chromatin extracted from rat neonatal cardiac myocytes that had been infected with adenovirus expressing siERRα or its backbone control (Super) and immunoprecipitated with IgG or antibodies against ERRα, PGC-1α, and Bcl3 as labeled. All analyses were as described above. α, anti.