The rat peroxisome-proliferator-activated receptor (PPAR) was expressed in insect cells and was shown to bind to a cognate PPAR response element (PPRE) from the acyl-CoA oxidase gene. Upon purification, PPAR was no longer able to bind DNA, although binding could be restored by addition of insect cell extracts. We investigated whether the retinoid X receptor (RXR) could supplement for this accessory activity. The rat RXRa cDNA was cloned and it was found that addition of in vitro-translated RXRa to purified PPAR facilitated binding of PPAR to a PPRE. Furthermore, an additional activity, which appeared to be distinct from rRXRa, was found in COS cell nuclear extracts that enabled binding of PPAR to a PPRE. Transient expression ofRXRa in CHO cells was found to be essential for the response of a chloramphenicol acetyltransferase reporter construct containing PPREs to activators of PPAR. These results raise the possibility of convergence of the PPAR and retinoid-dependent signaling pathways on promoters containing PPRE-like responsive elements.
The peroxisome proliferator activated receptor is a member of the steroid receptor gene superfamily, sharing amino acid sequence homology with other receptors and also showing similarities at the level of gene structure. This receptor is activated both by xenobiotic compounds that induce peroxisome proliferation and by fatty acids at physiological concentrations. Upon activation the receptor mediates transcription of responsive genes through binding to peroxisome proliferator response elements. These genes include those encoding peroxisomal enzymes and members of the cytochrome P450 family of drug metabolizing enzymes. It is therefore possible that the peroxisome proliferator activated receptor may play a crucial role in regulating lipid homeostasis.
A 1.0 kb region of the BPV-1 genome (the long control region, LCR), contains controls for transcription and the origin of replication. Transcription directed by the LCR is activated by the viral encoded E2 protein. To define the essential cis acting elements that are required to control transcription we have constructed a series of deletions throughout the LCR. We have identified three important domains in the LCR, two of which respond to E2. We have analysed the ability of small subcloned regions of the E2 responsive domains to act as enhancers in a heterologous assay system. This has led to the identification of five independent E2 responsive elements. We have shown that a fragment of only 38 base pairs is sufficient to respond to activation by E2. We also present evidence to suggest two types of E2 responsiveness that result in strong or weak activation. Strong response is correlated with the presence of the sequence 5'-ACCG/TNNNC/TCGGTGC-3' whereas weak response is correlated with the presence of a related sequence 5'-ACC(N)6GGT-3'. The contribution of these multiple elements to viral transcription is discussed.
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