Figure 2
From: Ribosomal transcription is regulated by PGC-1alpha and disturbed in Huntington’s disease
The rDNA association of PGC-1α is dependent on its activation state and influences promoter occupancy of the initiation complex. (2a) PGC-1α associates with the rDNA-promotor, gene internal sequences and the intergenic spacer only after stimulation with resveratrol. ChIP assay with untransfected HEK cells, kept under 3% pO2, treated with/without resveratrol (10 µM) for 3 h. Samples were normalized to input (native chromatin, positive control) N = 3; ****p < 0.0001; column: mean ± SD. (2b) Resveratrol treatment induces deacetylation of PGC-1α. Immunoprecipitation of lysates of flag-PGC-1α transfected cells treated without/with 10 µM resveratrol with anti-flag (M2) antibodies. The membrane was probed with acetyl-lysine antibody and, after stripping, with anti-flag antibody to detect flag PGC-1α. (2c) The effect of resveratrol on PGC-1α-association with the rDNA can completely be reversed by addition of the resveratrol antagonist nicotinamide (10 mM) for 3 h. ChIP assay. Samples were normalized to input (native chromatin, positive control). N = 3; ****p < 0.0001; column: mean ± SD. (2d) Promotor occupancy of Pol I and UBF is increased in HEK cells, treated with 10 µM resveratrol for 3h. HEK cells kept under 3% pO2 with/without resveratrol; ChIP assay, qPCR using promoter primer. Samples were normalized to input (native chromatin, positive control). N = 3; **p = 0.0022, ****p < 0.0001; column: mean ± SD. (2e) PGC-1α and UBF associate with the same rDNA sequences. ChIP-re-ChIP. Samples were normalized to input (native chromatin, positive control). N = 3, mean ± SD. (2f) Co-immunoprecipitation of PGC-1α with subsequent western blot analysis using UBF and RNA polymerase I antibodies (RPA135). Nuclear extracts of flag-tagged PGC-1α transfected HEK cells. N = 3. Anti-Flag ab for detection of PGC-1α 1:500. (Full-length blot see supplement)
