The molecular mechanism of transcriptional activation by the peroxisome proliferator activated-receptor alpha

Abstract

<p>The peroxisome proliferator-activated receptor (PPARα) is a member of the nuclear receptor superfamily, a growing class of transcriptional activators. Many of these proteins, including PPARα, function as intracellular receptors for hormones and upon the binding of agonist, activate the transcription of target genes. PPARα is an integral regulator of fatty acid metabolism and as such is activated by fatty acids resulting in the stimulation of the expression of genes involved in the release, cellular uptake, and β-oxidation of free fatty acids. The research presented in this thesis details the discovery and characterization of mechanistic aspects by which PPARα mediates the transcriptional activation of target genes. Transcriptional activators bind to specific DNA binding sites in the promoter region of target genes to effect changes in the rate of transcription. Included in the presented work are the discovery and the characterization of protein-DNA interactions that are involved the activation of transcription by PPAR. Firstly, this includes the identification of PPARs as the factors that mediated the activation of a target gene by free fatty acids. PPARs were shown to bind to their appurtenant DNA binding sites as a heterodimer with the retinoid X receptor (RXR). While the binding of this complex to DNA was required for PPAR mediated signalling, binding was not sufficient for transcriptional activation implying that the DNA binding site itself acted as an allosteric regulator. Secondly, evidence is presented that identifies two other nuclear receptors that bound to the same DNA binding sites as PPAR. These factors, the chicken ovalbumin upstream promoter-transcription factor 1 and the hepatocyte nuclear factor 4, modulated the activity of PPARα resulting in the abrogation and potentiation, respectively, of PPARα activity. Once bound to specific DNA sequences, transcriptional activators mediate their effects through a network of protein-protein interactions that ultimately controls the transcriptional process. Included in this thesis are studies that established that PPARα interacts with its DNA binding partner RXRα in vivo in the absence of exogenous agonist and DNA binding. This suggested that PPAR and RXR bound to DNA as a preformed heterodimer. To discover novel interacting partners for PPARα, the yeast two-hybrid screen was utilized. Two novel PPARα-protein interactions were identified. This included the discovery and cloning of a novel nuclear receptor, the liver X receptor that interacted with PPARα. Furthermore, an interaction was documented between PPARα and the receptor interacting protein 140, a putative mediator of the transcriptional activation activity of nuclear receptors. In both cases, the liver X receptor and receptor interacting protein 140, antagonized the transcriptional activation mediated by PPARα. These studies have succeeded in increasing the known repertoire of proteins that modulate and effect the activity of PPARα through protein-protein interactions. The findings presented in this thesis have contributed to the understanding of the network of protein-DNA and protein-protein interactions that both potentiate and antagonize the activity of PPARα. This has helped further our understanding of the mechanism by which fatty acids directly regulate the activity of PPARα and control the expression of target genes to elicit adaptive changes in metabolism.</p>