Cloning and Analyzing The Promoter of PPAR? / 生物化学与生物物理进展

ABSTRACT

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear hormone receptors belonging to the steroid receptor superfamily. Three PPAR isoforms, PPAR?, PPAR? (also known as PPAR?) and PPAR? have been found in the mouse. They can activate expression of many genes, including those involved in lipidmetabolism. PPAR? is ubiquitously expressed, but the level of expression differs markedly between different cell types. PPAR? is expressed in skeletal muscle at 10- and 50-fold higher levels compared with PPAR? and PPAR?, respectively. A role for PPAR? in skeletal muscle is to increase the genes expression with relation to oxidative metabolism. In order to determine the molecular mechanisms governing PPAR? gene expression in muscle, a 2 kb 5′ flanking region was cloned and analyzed. The DNA fragment is able to transcribe GFP in COS7 cells. Dual luciferase assay is used to quantify promoter activity. Deletion analysis of the 2 kb PPAR? promoter fragment in COS7 and NIH 3T3 cells shows that the proximal promoter sequence, nt -197 to +120, confers basal transcriptional activity of the mouse PPAR? gene. Computational analysis of putative cis-acting elements located within the ～2.0 kb mouse PPAR? 5′-flanking sequence was performed using the TRANSFAC database and MatInspector software and 4 potential MEF2A binding sites were found. And there is a potential binding site sharing 100% identity with positive element of MEF2A in the proximal promoter (nt -261). Co-transfection experiments of the PPAR? promoter reporter and pMEF2A expression plasmid (pMEF2A) showed that MEF2A significantly enhanced transcription activity of PPAR? promoter in NIH 3T3. Moreover, the enhancive effect depended on the concentration of plasmid pMEF2A transfected into cells. The results suggested that MEF2A may enhance transcription activity of the PPAR promoter in muscle cells.