Identification of an extended half-site motif required for the function of peroxisome proliferator-activated receptor alpha.
BACKGROUND
Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily and regulate many genes of the proteins involved in lipid metabolism, including peroxisomal acyl-CoA oxidase (AOX). Through heterodimerization with retinoid X receptors (RXRs), PPAR was believed to recognize the sequence elements consisting of two directly repeating 6-bp half-sites spaced by one nucleotide (DR-1), located in the regulatory regions of these genes.


RESULTS
Employing the peroxisome proliferator-responsive enhancer of the rat AOX gene, we analysed the minimal sequence requirements for enhancer activity and PPARalpha/RXRalpha binding. We found that the sequence just downstream of the DR-1 motif is indispensable for both functions. By a direct selection procedure of high-affinity binding sites from a random sequence pool, we identified a consensus sequence at the four positions next to DR-1. We also suggest that PPARalpha binds to the downstream half-site, whereas RXRalpha binds to the upstream half-site of the AOX DR-1.


CONCLUSIONS
An extended half-site of 10-bp, but not a simple 6-bp half-site, is required for the PPARalpha binding, upon heterodimer formation with RXRalpha. The binding polarity of PPARalpha/RXRalpha seems to be opposite to that of other RXR-involving heterodimers.
Osada
S
S
Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo, Japan.
Tsukamoto
T
T
Takiguchi
M
M
Mori
M
M
Osumi
T
T
D042962:Acyl-CoA Oxidase; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D016384:Consensus Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D008156:Luciferases / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D010088:Oxidoreductases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D047488:Retinoid X Receptors; D016415:Sequence Alignment; D017384:Sequence Deletion; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D010088:Oxidoreductases; D008156:Luciferases; C107176:peroxisomal acyl-CoA oxidase; D042962:Acyl-CoA Oxidase