Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Fishery College, Huazhong Agricultural University, Wuhan 430070, China.
Collaborative Innovation Center for Efficient and Health Production of Fisheries, Hunan University of Arts and Science, Changde 415000, China.
Int J Mol Sci. 2017 Nov 13;18(11):2405. doi: 10.3390/ijms18112405.
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.
肉碱棕榈酰转移酶 I（CPT I）是调节脂肪代谢和脂肪酸β氧化的关键酶。为了了解 和 基因的转录机制，我们分别克隆了草鱼（）的 和 启动子的 2695bp 和 2631bp 区域，并探讨了这些启动子的结构和功能特征。 有两个转录起始位点（TSS），而 只有一个 TSS。DNase I 足迹显示， 启动子富含 AT 且不含 TATA，通过起始因子（INR）独立机制介导基础转录。生物信息学分析表明，特异性蛋白 1（Sp1）和核因子 Y（NF-Y）在驱动 基因的基础表达中可能发挥重要作用。在 HepG2 和 HEK293 细胞中，逐步缺失分析表明，几个区域包含控制 和 基因转录的顺式元件。此外，一些转录因子，如甲状腺激素受体（TR）、肝细胞核因子 4（HNF4）和过氧化物酶体增殖物激活受体（PPAR）家族，都在 和 启动子上被鉴定出来。TRα 结合位点仅在 启动子上被鉴定出来，而 TRβ 结合位点仅在 启动子上被鉴定出来，这表明 和 的转录受到不同机制的调控。位点突变和电泳迁移率变动分析（EMSA）表明，非诺贝特诱导的 PPARα 激活不会与 启动子上预测的 PPARα 结合位点结合。此外，PPARα 不是调节 表达的唯一 PPAR 家族成员，PPARγ 也调节 表达。所有这些结果为鱼类 基因转录调控的机制提供了新的见解。
