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. 2010 May;38(9):2839-50.
doi: 10.1093/nar/gkq012. Epub 2010 Jan 27.

Profiling of promoter occupancy by PPARalpha in human hepatoma cells via ChIP-chip analysis

David L M van der Meer  1 , Tatjana Degenhardt, Sami Väisänen, Philip J de Groot, Merja Heinäniemi, Sacco C de Vries, Michael Müller, Carsten Carlberg, Sander Kersten
Affiliations

VSports最新版本 - Profiling of promoter occupancy by PPARalpha in human hepatoma cells via ChIP-chip analysis

David L M van der Meer et al. Nucleic Acids Res. 2010 May.

Abstract

The transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) is an important regulator of hepatic lipid metabolism VSports手机版. While PPARalpha is known to activate transcription of numerous genes, no comprehensive picture of PPARalpha binding to endogenous genes has yet been reported. To fill this gap, we performed Chromatin immunoprecipitation (ChIP)-chip in combination with transcriptional profiling on HepG2 human hepatoma cells treated with the PPARalpha agonist GW7647. We found that GW7647 increased PPARalpha binding to 4220 binding regions. GW7647-induced binding regions showed a bias around the transcription start site and most contained a predicted PPAR binding motif. Several genes known to be regulated by PPARalpha, such as ACOX1, SULT2A1, ACADL, CD36, IGFBP1 and G0S2, showed GW7647-induced PPARalpha binding to their promoter. A GW7647-induced PPARalpha-binding region was also assigned to SREBP-targets HMGCS1, HMGCR, FDFT1, SC4MOL, and LPIN1, expression of which was induced by GW7647, suggesting cross-talk between PPARalpha and SREBP signaling. Our data furthermore demonstrate interaction between PPARalpha and STAT transcription factors in PPARalpha-mediated transcriptional repression, and suggest interaction between PPARalpha and TBP, and PPARalpha and C/EBPalpha in PPARalpha-mediated transcriptional activation. Overall, our analysis leads to important new insights into the mechanisms and impact of transcriptional regulation by PPARalpha in human liver and highlight the importance of cross-talk with other transcription factors. .

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Figures

Figure 1.
Figure 1.
Mapping of PPARα binding regions enriched upon GW7647 treatment. (A) Positional distribution of all identified PPARα binding regions relative to TSSs of the nearest gene. (B) Identification of the genomic location of PPARα binding regions using PinkThing. The following classification criteria were used: distant (>25 kb), 5′ far (25–5 kb), 5′ near (5–0 kb), intron (intronic), exon (exonic), 3′ near (0–5 kb) and 3′ far (5–15 kb). (C) Enrichment of promoter regions in PPARα target genes. Enriched ChIP-chip signals were visualized using Affymetrix integrated genome browser. Coverage of promoter tiling array is indicated in red, repetitive sequences in black, and conserved sequence in blue. PPARα target genes SULT2A1, ACOX1, IGFBP1, ACADL, CD36 and G0S2 all show positive enrichment within promoter regions. No enrichment is observed in the promoter of ANGPTL4 as the known PPRE is present within the (non-covered) intron 3.
Figure 2.
Figure 2.
Overlap between GW7647-induced PPARα binding and GW7647-induced changes in expression. (A) Significant induction of PPARα targets by GW7647 treatment. (B) Number of genes significantly altered upon GW7647 treatment as determined by microarray analysis using criteria: fold change >1.2 and q-value <0.05. (C) Overlap between genes assigned to GW7647-induced PPARα binding regions and genes altered after treatment with GW7647 as determined by transcriptomics. (D) Percentage of GW7647-induced PPARα binding regions linked to either up- or down-regulated genes that contain at least one V$PERO site, as determined using Genomatix. Similar analysis was done for all GW7647-induced PPARα binding regions as well as a control set of promoter regions in the Genomatix promoter database with similar size range as the binding regions identified by ChIP-chip (1000–1500 bp).
Figure 3.
Figure 3.
Cross-talk between PPARα- and SREBP-dependent gene-regulation. (A) Enriched ChIP-chip signals for HMGCS1, HMGCR, LPIN1 and AGPAT9 genes were visualized using Affymetrix integrated genome browser. Coverage of promoter tiling array is indicated in red, repetitive sequences in black and conserved sequences in blue. (B) Gene expression changes after 6 h PPARα agonist treatment of five direct SREBP target genes and possible SREBP target gene AGPAT9. A GW7647-induced PPARα binding region was assigned to each of these genes. Significant differences are indicated with an asterisk (Student’s t-test, p < 0.05). (C) Transcriptional up-regulation of selected SREBP1 target genes involved in lipogenesis after 6 h PPARα agonist treatment represented as a heat map. (D) Enriched DNA binding of PPARα to promoter regions of LPIN1, AGPAT9 and HMGCR after 2 h GW7647 treatment, verified by ChIP-qPCR using primers designed within the binding region found by ChIP-Chip.
Figure 4.
Figure 4.
De novo motif analysis. GW7647-induced PPARα binding regions were screened for specific DNA motifs via de novo motif search using MEME. The binding regions of the 25 most significantly up-regulated genes assigned to GW7647-induced PPARα binding regions were analyzed. Significantly enriched motifs were compared with motif databases TRANSFAC as well as JASPAR with the use of STAMP. Similarity scores with known TF binding regions are expressed by E-values. One motif identified showed similarity to a PPARα motif within the TRANSFAC database, another motif identified showed similarity to the C/EBPα motif in the JASPAR database.
Figure 5.
Figure 5.
Enrichment of TF modules in PPARα binding regions. (A) The binding regions of the 25 most significantly up-regulated genes assigned to GW7647-induced PPARα binding regions were analyzed for TF modules using the Genomatix tool Frameworker. Two modules were identified: TBP-PERO and STAT-PERO in the binding regions linked to up- or down-regulated genes, respectively. (B) The modules TBP-PERO and STAT-PERO were scanned for the relative presence in all GW7647-induced PPARα binding regions located near transcriptional regulated as well as all human promoter regions present in the Genomatix database. A two-proportion z-test was used to analyze significant enrichment of modules. p-values values below 0.05 were considered significantly different. (C) Loss of DNA binding by STAT3 and STAT1 upon PPARα activation. HepG2 cells were treated with GW7647 for 2 h and ChIP performed using antibodies against STAT3, STAT1 and STAT6 with vehicle-treated HepG2 cells serving as control. Precipitated chromatin was subsequently amplified using primers around the predicted STAT-PERO site found in four genes that were linked to a GW7647-induced PPARα binding region and were down-regulated by GW7647. Significant differences are indicated with an asterisk (Student's; t-test, p<0.05).
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