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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: Biochim Biophys Acta. 2011 Oct 7;1819(1):1–15. doi: 10.1016/j.bbagrm.2011.09.006

Fig. 6. Identification of TNIP1 promoter element C as a functional PPRE.

Fig. 6

(A) Schematic of reporter constructs. Candidate PPREs (diagonal striped bars) A, B, and C were introduced as single or triple inserts in basal reporter construct. Stippled box, tk minimal promoter. LUC, 5′ end of luciferase coding region.

(B) TNIP1 promoter element C provides PPAR responsiveness. HeLa cells transfected with equal copy number luciferase reporter constructs with tk minimal promoter only or tk promoter ligated to TNIP1 distal promoter fragment −5206 ~ −4396, or single, or three times repeats of candidate elements A, B, or C, or a mini-gene version of the candidate PPREs in a contiguous stretch of sequence (see schematics in A). Transfections, expression and reporter constructs, vehicle (open bars) and ligands (solid bars), as in Figure 3. Normalized RLU’s are presented from representative experiment with signal from tk-Luc construct in the presence of vehicle set at 1; note breaks in y-axis and changes in scale. Fold increase in normalized RLU’s are presented with signal from the tk-Luc construct in absence of transfected nuclear receptor heterodimer, vehicle control, set at 1 (first open bar). Error bars are SD. Statistically significant differences indicated by ^, p<0.05, or *, p<0.01, from Student’s t-test for reporter activity without (−/−) versus with (+/+) expression of PPARγ/RXRα, within vehicle or ligand treatments. ns, not significant difference.

(C) TNIP1 promoter element C functions as an orientation-independent PPRE. HeLa cells transfected and cultured as in B. Normalized RLU’s are presented with signal from tk-Luc construct in presence of vehicle set at 1. Ligand for transfections with the PPARγ/RXRα heterodimer pair or PPARγ alone was troglitazone as in Figure 3. Ligand for RXRα alone transfection was 1μM 9-cis RA. Error bars are SEM. REV 3xC is a three times repeat of element C but in an orientation reverse that occurring in the native promoter. Statistically significant differences indicated by *, p<0.01, or #, p<0.001, from Student’s t-test for reporter activity without (−/−) versus with (+/+) expression of PPARγ/RXRα. ns, not significant difference. +, p<0.005 for troglitazone induction.

(D) Mutation of element C in the 6kb promoter construct reduces activation by PPAR. HeLa cells transfected and cultured as in B. Normalized RLU’s are presented with signal from the wild-type 6kb-Luc construct (wt-6kb) without transfected receptors, in presence of vehicle, set at 1. Ligands as in Figure 3E. Statistically significant differences indicated by #, p<0.001, from Student’s t-test for the 6kb-Luc construct with a mutated element C (mtC-6kb) compared against the corresponding condition for wt-6kb.