DNA binding preferences of PPAR alpha/RXR alpha heterodimers.

The regulatory elements mediating the transcriptional effects of the Peroxisome Proliferator Activated Receptor (PPAR)/Retinoid X Receptor heterodimers consist of a direct repeat of a variant of the consensus hexamer AGGTCA with an interspacing of 1 basepair (DR1). A binding site selection was performed to investigate whether any further constraints for PPAR/RXR binding to DR1 elements exist and/or whether other high affinity binding sites for these heterodimers can be identified. One half of the recovered sequences contained two hexamers related to the consensus halfsite organised as DR1, DR2, PAL0 or as DR3, in diminishing order of frequency. The other binding sites consisted of three hexamer repeats with the number of interspacing bases varying between 0 and 7. An element with three consecutive hexamer sequences each spaced by 1 basepair was most efficient in mediating the effects of peroxisome proliferators. The results indicate that the upstream flanking sequence of a DR1 differentially influences the binding of PPAR alpha/RXR alpha heterodimers and of RXR alpha homodimers.

Sequence requirements for high affinity retinoid X receptor-alpha homodimer binding.

To better delineate the sequence requirements for high affinity binding of retinoid X receptor alpha (RXR alpha) homodimers, a selection protocol was used starting from a random pool of oligonucleotides. All recovered sequences contained at least two hexamers related to the consensus sequence for the thyroid/retinoid subfamily of nuclear receptors, A/GGGTCA. These hexamers were most frequently organised as direct repeats with one interspacing base pair (DR1) and as palindromic repeats without interspacing base pairs (PAL0), the established configurations for RXR response elements (RXREs). However, DR2 and DR6 configurations also appeared to bind RXR alpha homodimers with high affinity, as did elements consisting of three hexamers. Reporters containing single copies of these elements conferred 9-cis retinoic acid responsiveness to cells cotransfected with an RXR alpha expressing plasmid. The upstream hexamer of all recovered sites was preferentially preceded by a G and its consensus was GGGTCA. Based on the composition of the selected DR1 RXREs, and the functional and mutational analysis, the optimal DR1 RXRE consists of an upstream hexamer starting with A or G and preceded by A or G. The interspacing base can be either G, A or T but not C. The affinity of RXR alpha homodimers for a DR1 element is strongly reduced when the final position is taken by a C. The results of the present investigation indicate that RXR alpha homodimers may have broader DNA binding specificities than currently believed. The biological relevance of these alternative RXREs will need to be corroborated by the identification of natural elements of this kind.

Antagonism of COUP-TF and PPAR alpha/RXR alpha on the activation of the malic enzyme gene promoter: modulation by 9-cis RA.

We previously demonstrated that heterodimers of the Peroxisome Proliferator Activated Receptor alpha (PPAR alpha) and the Retinoid X Receptor alpha (RXR alpha) stimulate malic enzyme gene transcription through a regulatory element in the promoter region (ME-PPRE). In this report, we show that the orphan nuclear receptor COUP-TF also displays affinity for the ME-PPRE and competes with PPAR alpha/RXR alpha for binding to this element. In transient transfections of a reporter driven by the MRE-PPRE in a heterologous or in the homologous promoter context, COUP-TF strongly antagonizes the transactivation by PPAR alpha RXR alpha in the absence of exogenously added ligands. Although 9-cis RA did not further enhance the transcriptional effects of the heterodimers activated by ciprofibrate, it greatly impaired the suppressive effects of COUP-TF on the ciprofibrate activated PPAR alpha/RXR alpha. We conclude that the antagonism by COUP-TF uncovers differential activation states of PPAR alpha/RXR alpha heterodimers in the absence and in the presence of 9-cis RA.

The peroxisome proliferator activated receptor regulates malic enzyme gene expression.

A new regulatory element for peroxisome proliferator activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers was found in the promoter of the malic enzyme gene. Similar to previously characterized peroxisome proliferator response elements (PPREs), it consists of a direct repeat of sequences related to the half-site consensus AGGTCA with an interspacing of 1 base pair. Specific binding of PPAR/RXR heterodimers to this element was demonstrated. Furthermore, this sequence conferred ciprofibrate responsiveness of a reporter through the homologous malic enzyme or heterologous thymidine kinase promoters. This PPRE presumably mediates the transcriptional effects of peroxisome proliferators on malic enzyme expression. The presence of a PPRE in the promoter of this lipogenic enzyme suggests a broader function for the PPAR in the regulation of lipid metabolism.

Peroxisome proliferators and T3 operate by way of distinct receptors.

Peroxisome proliferators and thyroid hormones have a number of common metabolic effects. The possibility that the signal transduction pathways of both groups of effectors converge at the receptor level was investigated. It was shown that T3, specifically bound to the rat thyroid beta-receptor, was not displaced to a significant extent by ciprofibrate or bezafibrate. No specific binding of T3 to the mouse peroxisome proliferator activated receptor could be demonstrated. In transactivation experiments peroxisome proliferators were unable to activate the thyroid receptor and T3 did not activate a chimeric receptor containing the ligand binding domain of the peroxisome proliferator activated receptor. It is concluded that peroxisome proliferators and thyroid hormone do not cross-react at the level of their nuclear receptors.