Analysis of the Regulatory Mechanism of Transcription by Acyl-CoA Oxidase Gene Enhancer

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1. To investigate the mechanism of induction of peroxisomal functions by peroxisome proliferators, we analyzed the enhancer of the gene for rat acyl-CoA oxidase, the key enzyme of the peroxisomal beta-oxidation system. By gene transfection to cultured animal cells from hepatic and non-hepatic origins, we found that both the direct repeat motif (DR-1) in the enhancer region and its downstream sequence are important for the transcriptional activation. These sequences were revealed to be necessary for the binding with the heterodimer of PPAR and RXR,nuclear receptors both involved in the activation of the gene.2. Roles of the DR-1 downstream sequence were extensively analyzed by transfection and protein binding assays using various mutant enhancer sequences. We found that the four nucleotide sequence next to the DR-1 motif is imortant for both the enhancer activity and PPAR/RXR binding. By selection of high-affinity binding sites from a random sequence pool, a consensus sequence for PPAR/RXR binding, A・G・A or T・T was identified. Furthermore, we showed that PPAR binds to the 3', whereas RXR to the 5'half-site of the acyl-CoA oxidase gene DR-1. This means that the extended half-site motif on the 3' side is required for binding of PPAR.The polarity of binding and requirement for an extended half-site are unique to PPAR,not observed for other heterodimer-forming nuclear receptors.3. We cloned three new proteins interacing with PPAR,by yeast two-hybrid system. One of them belongs to the nuclear hormone receptor superfamily, but lacks a Zn-finger DNA-binding domain at the N-terminus. This protein also interacts with other nuclear receptors, and thus this protein might modulates the functions of various nuclear receptors.4. We observed that PPAR and HNF-4 functionally competes with each other in the transcriptional regulation. We also found that the N-terminal domain of PPAR carries an independent gene activation function.