Michael Boergesen

Genome-Wide Profiling of Liver X Receptor, Retinoid X Receptor, and Peroxisome Proliferator-Activated Receptor α in Mouse Liver Reveals Extensive Sharing of Binding Sites

ABSTRACT The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.

Differential effects of environmental chemicals and food contaminants on adipogenesis, biomarker release and PPARγ activation

Eleven environmental relevant chemicals were investigated for their ability to affect adipogenesis in vitro, biomarker release from adipocytes and PPARα and γ activation. We found that butylparaben stimulated adipogenesis in 3T3-L1 adipocytes and increased release of leptin, adiponectin and resistin from the cells. Butylparaben activated PPARγ as well, which may be a mediator of the adipogenic effect. Polychlorinated biphenyl (PCB)153 also stimulate adipogenesis and biomarker release, but did not affect PPARs. The data indicates that PPARγ activating chemicals often stimulate adipocyte differentiation although PPARγ activation is neither a requirement nor a guarantee for stimulation. Four out of the eleven chemicals (bisphenol A, mono-ethylhexyl phthalate, butylparaben, PCB 153) caused increased adipogenesis. The release of adipocyte-secreted hormones was sometimes but not always correlated with the effect on adipocyte differentiation. Eight chemicals were able to cause increased leptin release. These findings strengthen the hypothesis that chemicals can interfere with pathways related to obesity development.

Peroxisome Proliferator-Activated Receptor γ and C/EBPα Synergistically Activate Key Metabolic Adipocyte Genes by Assisted Loading

ABSTRACT Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are key activators of adipogenesis. They mutually induce the expression of each other and have been reported to cooperate in activation of a few adipocyte genes. Recently, genome-wide profiling revealed a high degree of overlap between PPARγ and C/EBPα binding in adipocytes, suggesting that cooperativeness could be mediated through common binding sites. To directly investigate the interplay between PPARγ and C/EBPα at shared binding sites, we established a fibroblastic model system in which PPARγ and C/EBPα can be independently expressed. Using RNA sequencing, we demonstrate that coexpression of PPARγ and C/EBPα leads to synergistic activation of many key metabolic adipocyte genes. This is associated with extensive C/EBPα-mediated reprogramming of PPARγ binding and vice versa in the vicinity of these genes, as determined by chromatin immunoprecipitation combined with deep sequencing. Our results indicate that this is at least partly mediated by assisted loading involving chromatin remodeling directed by the leading factor. In conclusion, we report a novel mechanism by which the key adipogenic transcription factors, PPARγ and C/EBPα, cooperate in activation of the adipocyte gene program.

MED14 Tethers Mediator to the N-Terminal Domain of Peroxisome Proliferator-Activated Receptor γ and Is Required for Full Transcriptional Activity and Adipogenesis

ABSTRACT The Mediator subunit MED1/TRAP220/DRIP205/PBP interacts directly with many nuclear receptors and was long thought to be responsible for tethering Mediator to peroxisome proliferator-activated receptor (PPAR)-responsive promoters. However, it was demonstrated recently that PPARγ can recruit Mediator by MED1-independent mechanisms. Here, we show that target gene activation by ectopically expressed PPARγ and PPARα is independent of MED1. Consistent with this finding, recruitment of PPARγ, MED6, MED8, TATA box-binding protein (TBP), and RNA polymerase II (RNAPII) to the enhancer and proximal promoter of the PPARγ target gene Fabp4 is also independent of MED1. Using a small interfering RNA (siRNA)-based approach, we identify MED14 as a novel critical Mediator component for PPARγ-dependent transactivation, and we demonstrate that MED14 interacts directly with the N terminus of PPARγ in a ligand-independent manner. Interestingly, MED14 knockdown does not affect the recruitment of PPARγ, MED6, and MED8 to the Fabp4 enhancer but does reduce their occupancy of the Fabp4 proximal promoter. In agreement with the necessity of MED14 for PPARγ transcriptional activity, we show that knockdown of MED14 impairs adipogenesis of 3T3-L1 cells. Thus, MED14 constitutes a novel anchoring point between Mediator and the N-terminal domain of PPARγ that is necessary for functional PPARγ-mediated recruitment of Mediator and transactivation of PPARγ subtype-specific target genes.

PPARdelta is a fatty acid sensor that enhances mitochondrial oxidation in insulin-secreting cells and protects against fatty acid-induced dysfunction.

The peroxisome proliferator-activated receptor δ (PPARδ) is implicated in regulation of mitochondrial processes in a number of tissues, and PPARδ activation is associated with decreased susceptibility to ectopic lipid deposition and metabolic disease. Here, we show that PPARδ is the PPAR subtype expressed at the highest level in insulinoma cells and rat pancreatic islets. Furthermore, PPARδ displays high transcriptional activity and acts in pronounced synergy with retinoid-X-receptor (RXR). Interestingly, unsaturated fatty acids mimic the effects of synthetic PPARδ agonists. Using short hairpin RNA-mediated knockdown, we demonstrate that the ability of unsaturated fatty acids to stimulate fatty acid metabolism is dependent on PPARδ. Activation of PPARδ increases the fatty acid oxidation capacity in INS-1E β-cells, enhances glucose-stimulated insulin secretion (GSIS) from islets, and protects GSIS against adverse effects of prolonged fatty acid exposure. The presented results indicate that the nuclear receptor PPARδ is a fatty acid sensor that adapts β-cell mitochondrial function to long-term changes in unsaturated fatty acid levels. As maintenance of mitochondrial metabolism is essential to preserve β-cell function, these data indicate that dietary or pharmacological activation of PPARδ and RXR may be beneficial in the prevention of β-cell dysfunction.

ChREBP Mediates Glucose Repression of Peroxisome Proliferator-activated Receptor α Expression in Pancreatic β-Cells

Chronic exposure to elevated levels of glucose and fatty acids leads to dysfunction of pancreatic β-cells by mechanisms that are only partly understood. The transcription factor peroxisome proliferator-activated receptor α (PPARα) is an important regulator of genes involved in fatty acid metabolism and has been shown to protect against lipid-induced β-cell dysfunction. We and others have previously shown that expression of the PPARα gene in β-cells is rapidly repressed by glucose. Here we show that the PPARα gene is transcribed from five alternative transcription start sites, resulting in three alternative first exons that are spliced to exon 2. Expression of all PPARα transcripts is repressed by glucose both in insulinoma cells and in isolated pancreatic islets. The observation that the dynamics of glucose repression of PPARα transcription are very similar to those of glucose activation of target genes by the carbohydrate response element-binding protein (ChREBP) prompted us to investigate the potential role of ChREBP in the regulation of PPARα expression. We show that a constitutively active ChREBP lacking the N-terminal domain efficiently represses PPARα expression in insulinoma cells and in rodent and human islets. In addition, we demonstrate that siRNA-mediated knockdown of ChREBP abrogates glucose repression of PPARα expression as well as induction of well established ChREBP target genes in insulinoma cells. In conclusion, this work shows that ChREBP is a critical and direct mediator of glucose repression of PPARα gene expression in pancreatic β-cells, suggesting that ChREBP may be important for glucose suppression of the fatty acid oxidation capacity of β-cells.

Peroxisome Proliferator-Activated Receptor-α Is a Functional Target of p63 in Adult Human Keratinocytes

p63 is a master switch in the complex network of signaling pathways controlling the establishment and maintenance of stratified epithelia. We provide evidence that peroxisome proliferator-activated receptor-alpha (PPARalpha), a ligand-activated nuclear receptor that participates in the skin wound healing process, is a target of p63 in human keratinocytes. Silencing of p63 by RNA interference and transient transfections showed that p63 represses PPARalpha through a functional region of promoter B. Chromatin immunoprecipitation analyses indicate that p63 is bound to this region, in the absence of a recognizable p63-binding motif, suggesting that it acts through interactions with other transcription factors (TFs). Distinct PPARalpha transcripts are differentially regulated by p63, indicating a bimodal action in promoter and/or transcription start specification. PPARalpha repression is consistent with lack of expression in the interfollicular epidermis under physiological conditions. Furthermore, we show that PPARalpha is a negative regulator of DeltaNp63alpha levels and that it also binds to a functional region of the DeltaNp63 promoter that lacks PPRE motifs. Therefore, the reciprocal regulation is exerted either through binding to non-consensus sites or through interactions with other DNA-bound TFs. In conclusion, our data establish a link between two TFs intimately involved in the maintenance of skin homeostatic conditions.