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

Abstract

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 CPT Iα1b and CPT Iα2a genes, we cloned the 2695-bp and 2631-bp regions of CPT Iα1b and CPT Iα2a promoters of grass carp (Ctenopharyngodon idella), respectively, and explored the structure and functional characteristics of these promoters. CPT Iα1b had two transcription start sites (TSSs), while CPT Iα2a had only one TSS. DNase I foot printing showed that the CPT Iα1b 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 Iα2a gene. In HepG2 and HEK293 cells, progressive deletion analysis indicated that several regions contained cis-elements controlling the transcription of the CPT Iα1b and CPT Iα2a 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 Iα1b and CPT Iα2a promoters. The TRα binding sites were only identified on CPT Iα1b promoter, while TRβ binding sites were only identified on CPT Iα2a promoter, suggesting that the transcription of CPT Iα1b and CPT Iα2a 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 CPT I promoters. Additionally, PPARα was not the only member of PPAR family regulating CPT I expression, and PPARγ 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.

Functional Analysis of Promoters from Three Subtypes of the PI3K Family and Their Roles in the Regulation of Lipid Metabolism by Insulin in Yellow Catfish Pelteobagrus fulvidraco

“…In human, Yang et al [ 8 ] confirmed that PI3KCa was transcriptionally regulated through NF-κB pathway. PPARs are key transcriptional factors which mediate the regulation of many enzymes in lipid metabolism [ 31 , 32 ]. In the present study, we identify that the PPAR-RXR binding site (located at −1788/−1767 bp of PI3KC2b promoter) is a potential positive regulator in regulating PI3KC2b promoter activity from yellow catfish.…”

“…EMSA was performed to confirm the functional binding sites of HNF1, IK1 and FOXO1 according to the protocols described in our recent publications [ 32 ]. Proteins for EMSA were extracted from HepG2 cell lines.…”

Novel insights for SREBP-1 as a key transcription factor in regulating lipogenesis in a freshwater teleost, grass carp Ctenopharyngodon idella

“…We identified the 5 0 complementary DNA (cDNA) sequences and the transcription start sites of srebp-1, accα, scd1 and fas of grass carp using RNA ligase-mediated rapid amplification of 5 0 cDNA ends (RLM-5 0 RACE) method. The promoter cloning was performed based on the published draft genome of grass carp (18) , and the protocols followed these described in our recent studies (19) . Genomic DNA was extracted from grass carp tail fins using a commercial kit (Tissue DNA Kit; Omega).…”

“…For promoter luciferase assays, HepG2 cells were counted and seeded at a density of 1 × 10 5 in twenty-four well plates, then cultured and transfected as mentioned in our recent studies (19,23) . Briefly, to study the nSREBP-1-induced changes in promoter activities, we co-transfected 300 ng of nSREBP-1 plasmid or the same amount of pcDNA3.1(þ) plasmid (300 ng, control) with 500 ng of each of these luciferase reporter plasmids of srebp-1, accα, scd1 and fas promoters into HepG2 cells using Lipofectamine 2000 (Invitrogen) at 80-90 % confluence, respectively.…”

Functional Analysis of Promoters of Genes in Lipid Metabolism and Their Transcriptional Response to STAT3 under Leptin Signals

“…At present, studies on the promoters of lipid metabolism-related genes are scarce in fish. Recently, in our laboratory, Xu et al [ 17 ] analyzed the structure and function of CPT I promoter in grass carp, but no studies have been reported for the promoter structure and function of ACCα , FAS , and PPARγ in teleosts.…”