Figure 3. ERRα interacts with the mouse Sirt3 promoter in vitro and in vivo.
A, Electrophoretic mobility shift assay was executed using a biotin probe. Biotin-labeled double-stranded oligonucleotides were incubated with or without nuclear extract containing ERRα protein. Approximately 200-fold excess wild-type and ERRE-mutated unlabeled double-stranded oligonucleotides were used for competitive inhibition. B, Primary hepatocytes were isolated, cultured in 100 mm dishes, and infected with adenoviruses expressing ERRα-FLAG and/or PGC-1α, or GFP as a control. For ChIP, protein-DNA complexes were immunoprecipitated with anti-FLAG or control IgG antibody. The mSirt3 promoter region harboring the ERRE site was amplified by PCR. C, Primary hepatocytes were isolated from mouse liver, and chromatin was immunoprecipitated with anti-PGC-1α antibody. Normal IgG was used as control. The mSirt3 promoter region harboring the ERRE (proximal region) could be amplified by PCR. However, the distal region of the mSirt3 promoter, having no ERRE and used as a negative control, could not be amplified.