Selective modulation of promoter recruitment and transcriptional activity of PPARgamma

Abstract

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor regulated by the insulin-sensitizing thiazolidinediones (TZDs). We studied selective modulation of endogenous genes by PPARgamma ligands using microarray, RNA expression kinetics, and chromatin immunoprecipitation (ChIP) in 3T3-L1 adipocytes. We found over 300 genes that were significantly regulated the TZDs pioglitazone, rosiglitazone, and troglitazone. TZD-mediated expression profiles were unique but overlapping. Ninety-one genes were commonly regulated by all three ligands. TZD time course and dose-response studies revealed gene- and TZD-specific expression kinetics. PEPCK expression was induced rapidly but PDK4 expression was induced gradually. Troglitazone EC50 values for PEPCK, PDK4, and RGS2 regulation were greater than those for pioglitazone and rosiglitazone. TZDs differentially induced histone acetylation of and PPARgamma recruitment to target gene promoters. Selective modulation of PPARgamma by TZDs resulted in distinct expression profiles and transcription kinetics which may be due to differential promoter activation and chromatin remodeling of target genes.

Figure 1. Venn diagrams of TZD expression profiles

The total number of genes regulated by a particular TZD is shown next to the name. The number of genes uniquely regulated by a TZD is contained in the non-overlapping regions of each circle. The numbers of genes similarly regulated by two or three TZDs are contained in the overlapping regions of the circles. Replicates: pioglitazone, n=7; rosiglitazone and troglitazone, n=10 each.

Figure 2. Time course curves for TZD-regulated genes

Genes activated (A-C) and repressed (D&E) by pioglitazone (20μM, triangles), rosiglitazone (1μM, circles), and troglitazone (20μM, squares). Data are expressed as a percent of the 24 hr mRNA expression response for each TZD, averages ± SE. n=4-6 per condition.

Figure 3. Dose response curves for TZD-regulated genes

A-C. Gene-specific regulation. *ANOVA, p<0.05 PDK4 vs PEPCK and CAP. D-G. SPPARM effects. Max doses: pioglitazone (20μM, triangles), rosiglitazone (1μM, circles), and troglitazone (20μM, squares). *ANOVA, p<0.05 troglitazone vs pioglitazone and rosiglitazone. Data are expressed as a percent of the max dose mRNA expression response for each TZD, averages ± SE. n=2-4 per condition.

Figure 4. ChIP studies of endogenous promoters

A. Representative AQP7 and FATP triplicate promoter PCR products from input DNA and IP samples. Antibodies: IgG – control, PPARγ, AcH4 – acetylated histone H4. B. AcH4 ChIP studies of PEPCK, AQP7, and FATP promoters. C&D. PPARγ ChIP studies of PEPCK and FATP promoters, respectively. ChIP data are averages ± SE from two independent experiments, each done in duplicate. D – DMSO control, P – pioglitazone, R – rosiglitazone, T – troglitazone. * p<0.05 vs DMSO control, ** p<0.05 vs rosiglitazone, # p<0.05 vs pioglitazone and rosiglitazone.