Figure 9

Validation of novel PPAR target genes on human chromosome 19. (a) Real-time quantitative PCR was used to determine the inducibility of the mRNA expression of the indicated eight PPAR target genes, relative to the control gene RPLP0, in HepG2 cells. The cells were stimulated for 2, 4 and 6 h with 100 nM GW7647. (b) An overview of the genomic organization of the human LASS1 gene; 10 kB upstream and downstream of the TSS are shown. Putative REs were identified by in silico screening and the calculated binding strengths of the PPAR subtypes are represented by columns in reference to a consensus DR1-type PPRE. All putative PPRE sequences are available on request. (c) Reporter gene assays were performed with extracts from HepG2 cells that were transiently transfected with luciferase reporter constructs containing genomic regions of the LASS1 gene together with empty expression vector (endogenous PPAR) or the indicated expression vectors for PPARα, PPARγ and PPARβ/δ. Cells were then treated for 16 h with solvent or PPAR subtype-specific ligands. Relative luciferase activity was determined and normalized to the activity of empty cloning vector control co-transfected with empty expression vector. (d) Chromatin was extracted from HepG2 cells that had been treated with solvent or for 120 minutes with 100 nM GW7647. The association of PPARα, RXRα and pPol II was monitored by ChIP assays with respective antibodies on three genomic regions of the LASS1 gene. Real-time quantitative PCR was performed on chromatin templates and fold change of antibody-precipitated template in relation to IgG-precipitated specificity control template was calculated. Columns in (a, c, d) represent means of at least three experiments and bars indicate standard deviations. Two-tailed Student's t-tests were performed to determine the significance (*p < 0.05, **p < 0.01).