Structure and Functional Analysis of Promoters from Two Liver Isoforms of CPT I in Grass Carp Ctenopharyngodon idella.

Carnitine palmitoyltransferase I (CPT I) is a key enzyme involved in the regulation of lipid metabolism and fatty acid β-oxidation. To understand the transcriptional mechanism of and genes, we cloned the 2695-bp and 2631-bp regions of and promoters of grass carp (), respectively, and explored the structure and functional characteristics of these promoters. had two transcription start sites (TSSs), while had only one TSS. DNase I foot printing showed that the promoter was AT-rich and TATA-less, and mediated basal transcription through an initiator (INR)-independent mechanism. Bioinformatics analysis indicated that specificity protein 1 (Sp1) and nuclear factor Y (NF-Y) played potential important roles in driving basal expression of gene. In HepG2 and HEK293 cells, progressive deletion analysis indicated that several regions contained cis-elements controlling the transcription of the and genes. Moreover, some transcription factors, such as thyroid hormone receptor (TR), hepatocyte nuclear factor 4 (HNF4) and peroxisome proliferator-activated receptor (PPAR) family, were all identified on the and promoters. The TRα binding sites were only identified on promoter, while TRβ binding sites were only identified on promoter, suggesting that the transcription of and was regulated by a different mechanism. Site-mutation and electrophoretic mobility-shift assay (EMSA) revealed that fenofibrate-induced PPARα activation did not bind with predicted PPARα binding sites of promoters. Additionally, PPARα was not the only member of PPAR family regulating expression, and PPARγ also regulated the expression. All of these results provided new insights into the mechanisms for transcriptional regulation of genes in fish.