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Int. J. Mol. Sci. 2017, 18(11), 2405; https://doi.org/10.3390/ijms18112405

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

1
Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Fishery College, Huazhong Agricultural University, Wuhan 430070, China
2
Collaborative Innovation Center for Efficient and Health Production of Fisheries, Hunan University of Arts and Science, Changde 415000, China
*
Author to whom correspondence should be addressed.
Received: 7 October 2017 / Revised: 1 November 2017 / Accepted: 10 November 2017 / Published: 13 November 2017
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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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. View Full-Text
Keywords: Ctenopharyngodon idella; carnitine palmitoyltransferase I; promoters; peroxisome proliferator-activated receptor; transcriptional regulation Ctenopharyngodon idella; carnitine palmitoyltransferase I; promoters; peroxisome proliferator-activated receptor; transcriptional regulation
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Xu, Y.-H.; Luo, Z.; Wu, K.; Fan, Y.-F.; You, W.-J.; Zhang, L.-H. Structure and Functional Analysis of Promoters from Two Liver Isoforms of CPT I in Grass Carp Ctenopharyngodon idella. Int. J. Mol. Sci. 2017, 18, 2405.

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