Immunoselection and characterization of a human genomic PPAR binding fragment located within POTE genes.
Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors and belong to the nuclear hormone receptor superfamily. They form heterodimers with retinoid X receptor (RXR) and bind to specific PPAR-response elements. To identify novel PPAR target genes, we developed an affinity method to isolate human genomic fragments containing binding sites for PPARs. For this, an antibody raised against all PPAR subtypes was used. Immunoselected fragments were amplified and sequenced. One of them, ISF1029, was mapped by BLAT and BLAST searches on different human chromosomes, downstream of several POTE genes. ISF1029 contained three hexamers strongly related to the AGGTCA motif organized according to a DR0/3 motif. The latter was found to bind to PPARalpha in gel mobility shift and supershift assays and to exhibit a downregulation potentiality in transfection experiments under clofibrate treatment. POTE genes were shown to be highly expressed in human Caco-2 colorectal adenocarcinoma cells and downregulated by fenofibrate and 9-cis-retinoic acid, as attested by RT-PCR assays. Microarray analysis confirmed and extended to the human T98-G glioblastoma cells, the downregulation of several POTE genes expression by Wy-14,643, a potent PPARalpha activator. Our data provide new insights about the pleiotropic action of PPARs.
Murad
Hossam
H
EA3446 Proliférateurs de Peroxysomes Université Henri Poincaré-Nancy I, Faculté des Sciences, BP239, 54506 Vandoeuvre-les-Nancy, France.
Collet
Philippe
P
Brunner
Emilie
E
Schohn
Hervé
H
Bécuwe
Philippe
P
Devignes
Marie-Dominique
MD
Dauça
Michel
M
Domenjoud
Lionel
L
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015139:Blotting, Southern; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D004247:DNA / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D015894:Genome, Human; D045325:HCT116 Cells; D006801:Humans; D047468:Immunoprecipitation; D008957:Models, Genetic; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D047492:Peroxisome Proliferator-Activated Receptors / Q000819:agonists / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D047492:Peroxisome Proliferator-Activated Receptors; D004247:DNA