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. Author manuscript; available in PMC: 2012 Nov 2.
Published in final edited form as: Cell Metab. 2011 Nov 2;14(5):598–611. doi: 10.1016/j.cmet.2011.10.001

Figure 6.

Figure 6

The role of RXR in PPARα/Sirt1-mediated ERRE suppression. (A) PPARα and Sirt1 cooperatively suppress ERR target gene promoters. (B) The ERRE is an important interface for PPARα/Sirt1-mediated gene suppression. (A–B) Schematic representations of each promoter are shown in these panels. The common hexad motifs of HREs are indicated by a red line (AGGTCA and AGGACA), blue line (one nucleotide redundancy in the last 3 letters from AGGTCA or AGGACA) or black line (ERRE mutant: TCGGAATCA). (C) Sirt1 is a crucial partner for PPARα-induced ERRE suppression. (D) Transcriptional activation of PPARα is not essential for ERRE suppression. Schematic representation of PPARα and PPARαΔAF2 are shown. AF1: Activation function 1, DBD: DNA binding domain, LBD: ligand binding domain, AF2: activation function 2. HA-Sirt1 was co-immunoprecipitated with both Myc-PPARα and Myc-PPARαΔAF2. (E) PGC-1α fails to normalize PPARα/Sirt1-mediated ERRE suppression. (F) RXR is not necessary for interaction between PPARα and Sirt1. RXR was removed from heart lysates by immunodeprivation. PPARα was then immunoprecipitated with anti-Flag-antibody. (G) The effect of an RXRα mutant lacking the DNA binding domain (RXRα(ΔN)) on PPARα-induced PPRE activation and ERRE suppression. (H) The effect of full-length RXRα on PPARα-induced PPRE activation and ERRE suppression. (I) The dosage effect of PPRE-reporter on PPARα-induced reporter activity. The indicated amounts of pPPRE-luc reporter plasmids were transfected together with PPARα and RXRα expression vectors (0.3 μg). (J) The dosage effect of PPRE-reporter on PPARα/Sirt1-mediated transcriptional regulation. The mean value for activity with PPARα expression alone was expressed as 1. (K) Knockdown of Sirt1 enhances PPARα-induced PPRE activation. (L) RXRα counteracts PPARα/Sirt1-mediated suppression of PPRE reporter activity. (A–F and G–L) Luciferase assays were performed after 1 (A–B, D–E, and G–L) and 3 days (C and K) of transduction and transfection with the indicated constructs (N=6–12). Error bars represent S. E. M. (M) Schematic representation of the data shown in this figure. PPARα-induced PPRE activation is enhanced by RXRα but prevented by RXRα(ΔN). In contrast, PPARα/Sirt1-mediated ERRE suppression is not affected by RXRα or RXRα(ΔN) (Left). When RXR does not cover the half core site of the PPRE, the PPARα/Sirt1 complex suppresses transcription through the single hexad unit within the PPRE (Right).