Characterization of the human PPARalpha promoter: identification of a functional nuclear receptor response element.
PPARalpha is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. The factors regulating human PPARalpha (hPPARalpha) gene expression remain largely unexplored. To study the mechanisms controlling hPPARalpha expression, the hPPARalpha gene promoter was identified and characterized. First, an alternatively spliced exon within the 5'-untranslated region of the hPPARalpha gene was identified by RT-PCR. Next, the transcription start site was mapped and the hPPARalpha gene promoter was cloned and functionally analyzed. Because PPARalpha levels are elevated in tissues expressing the hepatocyte nuclear factor-4 (HNF4), such as liver, the regulation of hPPARalpha by HNF4 was examined. Transient transfections in HepG2 and Cos cells showed that HNF4 enhances hPPARalpha promoter activity. 5'-Deletion and mutation analysis of the hPPARalpha promoter identified a regulatory element (RE) consisting of a degenerate hexamer repeat with a single nucleotide spacer (direct repeat 1), termed alphaHNF4-RE. Gel shift assays demonstrated that HNF4 binds to this alphaHNF4-RE. Furthermore, HNF4 increased the activity of a heterologous promoter driven by two copies of the alphaHNF4-RE. The nuclear receptor COUP-TFII also bound this site and down-regulated basal as well as HNF4-induced hPPARalpha promoter activity. Finally, PPARalpha was shown to bind the alphaHNF4-RE, leading to an induction of PPARalpha expression in hepatocytes. In summary, the organization of the 5'-flanking and untranslated region of the hPPARalpha gene was characterized and the hPPARalpha promoter region has been identified. Furthermore, these data demonstrate that the hPPARalpha gene is regulated by nuclear receptors, such as HNF-4, COUP-TFII, and PPARalpha.
Pineda Torra
Inés
I
U.545 Institut National de la Santé et de la Recherche Médicale, Département d'Athérosclérose, Institut Pasteur de Lille, 59019 Lille, France.
Jamshidi
Yalda
Y
Flavell
David M
DM
Fruchart
Jean-Charles
JC
Staels
Bart
B
D020121:5' Untranslated Regions; D017398:Alternative Splicing; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D019556:COS Cells; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D005091:Exons; D018197:Hepatoblastoma; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D020121:5' Untranslated Regions; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors