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A map of the PPARα transcription regulatory network for primary human hepatocytes

ABSTRACT: Nuclear receptor activation in liver leads to coordinated alteration of the expression of multiple gene products with attendant phenotypic changes of hepatocytes. Peroxisome proliferators including endogenous fatty acids, environmental chemicals, and drugs induce a multi-enzyme metabolic response that affects lipid and fatty acid processing. We studied the signaling network for the peroxisome proliferator-associated receptor alpha (PPARα) in primary human hepatocytes using the selective PPARα ligand, GW7647. We measured gene expression over multiple concentrations and times and conducted ChIP-seq studies at 2 and 24 hours to assess genomic binding of PPARα. Over all treatments there were 192 genes differentially expressed. Of these only 51% showed evidence of PPARα binding – either directly at PPARα response elements or via alternative mechanisms. Almost half of regulated genes had no PPARα binding. We then developed two novel bioinformatics methods to visualize the dose-dependent activation of both the transcription factor circuitry for PPARα and the downstream metabolic network in relation to functional annotation categories. Available databases identified several key transcription factors involved with the non-genomic targets after GW7647 treatment, including SP1, STAT1, ETS1, ERα, and HNF4α. The linkage from PPARα binding through gene expression likely requires intermediate protein kinases to activate these transcription factors. We found enrichment of functional annotation categories for organic acid metabolism and cell lipid metabolism among the differentially expressed genes. Lipid transport processes showed enrichment at the highest concentration of GW7647 (10μM). While our strategy for mapping transcriptional networks is evolving, these approaches are necessary in moving from toxicogenomic methods that derive signatures of activity to methods that establish pathway structure, showing the coordination of the activated nuclear receptor with other signaling pathways.

ORGANISM(S): Homo sapiens

PROVIDER: GSE53399 | GEO | 2013/12/18

SECONDARY ACCESSION(S): PRJNA231926

REPOSITORIES: GEO

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Project description:Nuclear receptor activation in liver leads to coordinated alteration of the expression of multiple gene products with attendant phenotypic changes of hepatocytes. Peroxisome proliferators including endogenous fatty acids, environmental chemicals, and drugs induce a multi-enzyme metabolic response that affects lipid and fatty acid processing. We studied the signaling network for the peroxisome proliferator-associated receptor alpha (PPARα) in primary human hepatocytes using the selective PPARα ligand, GW7647. We measured gene expression over multiple concentrations and times and conducted ChIP-seq studies at 2 and 24 hours to assess genomic binding of PPARα. Over all treatments there were 192 genes differentially expressed. Of these only 51% showed evidence of PPARα binding – either directly at PPARα response elements or via alternative mechanisms. Almost half of regulated genes had no PPARα binding. We then developed two novel bioinformatics methods to visualize the dose-dependent activation of both the transcription factor circuitry for PPARα and the downstream metabolic network in relation to functional annotation categories. Available databases identified several key transcription factors involved with the non-genomic targets after GW7647 treatment, including SP1, STAT1, ETS1, ERα, and HNF4α. The linkage from PPARα binding through gene expression likely requires intermediate protein kinases to activate these transcription factors. We found enrichment of functional annotation categories for organic acid metabolism and cell lipid metabolism among the differentially expressed genes. Lipid transport processes showed enrichment at the highest concentration of GW7647 (10μM). While our strategy for mapping transcriptional networks is evolving, these approaches are necessary in moving from toxicogenomic methods that derive signatures of activity to methods that establish pathway structure, showing the coordination of the activated nuclear receptor with other signaling pathways. Primary hepatocytes from four donors were exposed to 0, 0.001, 0.01, 0.1, 1.0, or 10.0μM GW7647 for 2, 6, 12, 24, or 72 hours.

Project description:The transcription factor Peroxisome Proliferator-Activated Receptor M-NM-1 (PPARM-NM-1) is an important regulator of hepatic lipid metabolism. While PPARM-NM-1 is known to activate transcription of numerous genes, no comprehensive picture of PPARM-NM-1 binding to endogenous genes has yet been reported. To fill this gap, we performed ChIP-chip in combination with transcriptional profiling on HepG2 human hepatoma cells treated with the PPARM-NM-1 agonist GW7647. We found that GW7647 increased PPARM-NM-1 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 PPARM-NM-1, such as ACOX1, SULT2A1, ACADL, CD36, IGFBP1 and G0S2, showed GW7647-induced PPARM-NM-1 binding to their promoter. A GW7647-induced PPARM-NM-1-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 PPARM-NM-1 and SREBP signaling. Our data furthermore demonstrate interaction between PPARM-NM-1 and STAT transcription factors in PPARM-NM-1-mediated transcriptional repression, and suggest interaction between PPARM-NM-1 and TBP and C/EBPM-NM-1 in PPARM-NM-1-mediated transcriptional activation. Overall, our analysis leads to important new insights into the mechanisms and impact of transcriptional regulation by PPARM-NM-1 in human liver and highlight the importance of cross-talk with other transcription factors. HepG2 cells were grown in phenol red-free DulbeccoM-bM-^@M-^Ys modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mg/ml streptomycin and 100 U/ml penicillin. Cells were split the day before experiments. Cells were kept at 37 M-BM-0C and 5% CO2. The following day, cells were treated with either 100 nM of the PPARM-NM-1 agonist GW7647 or control vehicle (DMSO). Cells used for gene expression analysis were harvested after 6 h or 24 h of GW7647 treatment. This submission represents the gene expression component of the study.

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A map of the PPARα transcription regulatory network for primary human hepatocytes

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