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 beta-oxidation. To understand the transcriptional mechanism of CPT I alpha 1b and CPT I alpha 2a genes, we cloned the 2695-bp and 2631-bp regions of CPT I alpha 1b and CPT I alpha 2a promoters of grass carp (Ctenopharyngodon idella), respectively, and explored the structure and functional characteristics of these promoters. CPT I alpha 1b had two transcription start sites (TSSs), while CPT I alpha 2a had only one TSS. DNase I foot printing showed that the CPT I alpha 1b 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 CPT I alpha 2a gene. In HepG2 and HEK293 cells, progressive deletion analysis indicated that several regions contained cis-elements controlling the transcription of the CPT I alpha 1b and CPT I alpha 2a 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 CPT I alpha 1b and CPT I alpha 2a promoters. The TR binding sites were only identified on CPT I alpha 1b promoter, while TR binding sites were only identified on CPT I alpha 2a promoter, suggesting that the transcription of CPT I alpha 1b and CPT I alpha 2a was regulated by a different mechanism. Site-mutation and electrophoretic mobility-shift assay (EMSA) revealed that fenofibrate-induced PPAR activation did not bind with predicted PPAR binding sites of CPT I promoters. Additionally, PPAR was not the only member of PPAR family regulating CPT I expression, and PPAR also regulated the CPT I expression. All of these results provided new insights into the mechanisms for transcriptional regulation of CPT I genes in fish.