Tissue expression analysis, cloning, and characterization of the 5′-regulatory region of the bovine fatty acid binding protein 4 gene1

Li, A.; Zhao, Zhidong; Zhang, Y.; Fu, Changzhen; Wang, M.; Liu, Zan

doi:10.2527/jas.2015-9378

Cited by 14 publications

(11 citation statements)

References 38 publications

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“…In mammals, the core promoter has generally been identified near the transcription initiation site (28)(29) . For example, the core promoter of cattle (Bos taurus) fabp4 was present in the region of 272 bp upstream from the initiation codon (30) . The distinctive location of the T. ovatus fabp4 core promoter region indicated that there may be distinct regulatory mechanisms of fabp4 between T. ovatus and mammals.…”

Section: Discussionmentioning

confidence: 99%

PPARγregulatesfabp4expression to increase DHA content in golden pompano (Trachinotus ovatus) hepatocytes

et al. 2021

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N-3 long chain (≥C20) polyunsaturated fatty acids (LC-PUFA) are vital fatty acids for fish and humans. As a main source of n-3 LC-PUFA for human consumers, the n-3 LC-PUFA content of farmed fish is important. Previously, we identified fatty acid- binding protein (fabp)-4 as a candidate gene for regulating the n-3 LC-PUFA content. Herein, we further assessed the role of fabp4 in this process. First, a 2059 bp promoter sequence of fabp4 in Trachinotus ovatus was cloned and, using progressive deletion, determined -2006 bp to -1521 bp to be the core promoter sequence. The potential peroxisome proliferation activator (PPAR)-γ binding sites were predicted to occur in this region. A luciferase reporter assay showed that the promoter activity of fabp4 decreased following mutation of the PPARγ binding site and that PPARγ increased the fabp4 promoter activity in a dose-dependent manner, implying that T. ovatus fabp4 is a target of PPARγ. The overexpression of fabp4 or PPARγ increased the docosahexaenoic acid (DHA) content in hepatocytes, whereas suppression of their expression diminished this effect, suggesting that both fabp4 and PPARγ play an active role in regulating DHA content. Moreover, the inhibition of fabp4 attenuated the increase in PPARγ-mediated DHA content, and the overexpression of fabp4 alleviated this effect. Collectively, our findings indicated that fabp4, which is controlled by PPARγ, plays an important role in DHA content regulation. The new regulation axis can be considered a promising novel target for increasing the n-3 LC-PUFA content in T. ovatus.

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Section: Discussionmentioning

confidence: 99%

PPARγregulatesfabp4expression to increase DHA content in golden pompano (Trachinotus ovatus) hepatocytes

et al. 2021

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“…After differentiation of the C2C12 myoblasts into myotubes for 7 days, we prepared nuclear protein extracts as previously described [ 21 , 23 ]. We incubated probes of a 200 fmol of 5'-biotin labeled MYOG (or C/EBPß) with 10μg nuclear extracts of C2C12 myotubes (or 10μg3T3-L1nuclear extracts), 2μl 10×binding buffer, 1μl 50% Glycerol, 1μl MgCl2 and 1μl poly (dI.dC) in a volume of 20 μl.…”

Section: Methodsmentioning

confidence: 99%

Molecular Characterization and Transcriptional Regulation Analysis of the Bovine PDHB Gene

Zhang

Zhao

et al. 2016

PLoS ONE

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The pyruvate dehydrogenase beta subunit (PDHB) is a subunit of pyruvate dehydrogenase (E1), which catalyzes pyruvate into acetyl-CoA and provides a linkage between the tricarboxylic acid cycle (TCA) and the glycolysis pathway. Previous studies demonstrated PDHB to be positively related to the intramuscular fat (IMF) content. However, the transcriptional regulation of PDHB remains unclear. In our present study, the cDNA of bovine PDHB was cloned and the genomic structure was analyzed. The phylogenetic tree showed bovine PDHB to be closely related to goat and sheep, and least related to chicken. Spatial expression pattern analysis revealed the products of bovine PDHB to be widely expressed with the highest level in the fat of testis. To understand the transcriptional regulation of bovine PDHB, 1899 base pairs (bp) of the 5’-regulatory region was cloned. Sequence analysis neither found consensus TATA-box nor CCAAT-box in the 5’-flanking region of bovine PDHB. However, a CpG island was predicted from nucleotides -284 to +117. Serial deletion constructs of the 5’-flanking region, evaluated in dual-luciferase reporter assay, revealed the core promoter to be located 490bp upstream from the transcription initiation site (+1). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) in combination with asite-directed mutation experiment indicated both myogenin (MYOG) and the CCAAT/enhancer-binding protein beta (C/EBPß) to be important transcription factors for bovine PDHB in skeletal muscle cells and adipocytes. Our results provide an important basis for further investigation of the bovine PDHB function and regulation in cattle.

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“…The pGL3-basic vector was used as the external control. At 5 h after transfection, the media was replaced by DMEM with 2 % horse serum (HS) (Gibco-Invitrogen, Carlsbad, CA, USA) to induce differentiation of C2C12 myoblasts to myotubes for 40 h. At 45 h after transfection, the cells were lysed and analysed with a dual-luciferase reporter assay according to the manufacturer's instructions [25,26].…”

Section: Real-time Pcr Analysis Of Spatial Expression Patternmentioning

confidence: 99%

“…The PCR primers (FABP3-GSP1 and FABP3-GSP2, Supplemental Table 1) were adopted to acquire the 5 0 -end of the FABP3 gene. Reaction conditions were performed in the first PCR as described previously [25,26]. The second PCR template was a 20-fold dilution of the first PCR products.…”

Section: Molecular Cloning and Sequence Analysismentioning

confidence: 99%

Tissue expression analysis, cloning and characterization of the 5′-regulatory region of the bovine FABP3 gene

Wang

et al. 2016

Mol Biol Rep

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Fatty acid binding protein 3 (FABP3) is a member of the FABP family which bind fatty acids and have an important role in fatty acid metabolism. A large number of studies have shown that the genetic polymorphisms of FABP3 are positively correlated with intramuscular fat (IMF) content in domestic animals, however, the function and transcriptional characteristics of FABP3 in cattle remain unclear. Real-time PCR analysis revealed that bovine FABP3 was highly expressed in cardiac tissue. The 5'-regulatory region of bovine FABP3 was cloned and its transcription initiation sites were identified. Sequence analysis showed that many transcriptional factor binding sites including TATA-box and CCAAT-box were present on the 5'-flanking region of bovine FABP3, and four CpG islands were found on nucleotides from -891 to +118. Seven serial deletion constructs of the 5'-regulatory region evaluated in dual-luciferase reporter assay indicated that its core promoter was 384 base pairs upstream from the transcription initiation site. The transcriptional factor binding sites RXRα, KLF15, CREB and Sp1 were conserved in the core promoter of cattle, sheep, pigs and dogs. These results provide further understanding of the function and regulation mechanism of bovine FABP3.

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Tissue expression analysis, cloning, and characterization of the 5′-regulatory region of the bovine fatty acid binding protein 4 gene1

Cited by 14 publications

References 38 publications

PPARγregulatesfabp4expression to increase DHA content in golden pompano (Trachinotus ovatus) hepatocytes

PPARγregulatesfabp4expression to increase DHA content in golden pompano (Trachinotus ovatus) hepatocytes

Molecular Characterization and Transcriptional Regulation Analysis of the Bovine PDHB Gene

Tissue expression analysis, cloning and characterization of the 5′-regulatory region of the bovine FABP3 gene

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