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Transcriptional regulation of the human, porcine and bovine OCTN2 gene by PPARα via a conserved PPRE located in intron 1.

Luo H, Zhang Y, Guo H, Zhang L, Li X, Ringseis R, Wen G, Hui D, Liang A, Eder K, He D - BMC Genet. (2014)

Bottom Line: Using positional cloning and reporter gene assays we identified a functional PPRE, each in the intron 1 of the porcine, bovine, and human OCTN2 gene.Gel shift assay confirmed binding of PPARα to this PPRE in the porcine, bovine, and the human OCTN2 gene.This suggests that regulation of genes involved in carnitine uptake by PPARα is highly conserved across species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Animal Husbandry and Veterinary Medicine, Shanxi Provincial Academy of Agricultural Sciences, Taiyuan, 030031, P. R. China. lhd2638@126.com.

ABSTRACT

Background: The novel organic cation transporter 2 (OCTN2) is the physiologically most important carnitine transporter in tissues and is responsible for carnitine absorption in the intestine, carnitine reabsorption in the kidney and distribution of carnitine between tissues. Genetic studies clearly demonstrated that the mouse OCTN2 gene is directly regulated by peroxisome proliferator-activated receptor α (PPARα). Despite its well conserved role as an important regulator of lipid catabolism in general, the specific genes under control of PPARα within each lipid metabolic pathway were shown to differ between species and it is currently unknown whether the OCTN2 gene is also a PPARα target gene in pig, cattle, and human. In the present study we examined the hypothesis that the porcine, bovine, and human OCTN2 gene are also PPARα target genes.

Results: Using positional cloning and reporter gene assays we identified a functional PPRE, each in the intron 1 of the porcine, bovine, and human OCTN2 gene. Gel shift assay confirmed binding of PPARα to this PPRE in the porcine, bovine, and the human OCTN2 gene.

Conclusions: The results of the present study show that the porcine, bovine, and human OCTN2 gene, like the mouse OCTN2 gene, is directly regulated by PPARα. This suggests that regulation of genes involved in carnitine uptake by PPARα is highly conserved across species.

Show MeSH
Binding ofin vitrotranslated mouse PPARα/RXRα to the PPRE in the intron 1 of porcine, bovine and human OCTN2 gene. EMSA was performed using DIG-labeled oligonucleotides corresponding to the wild-type and the mutated PPRE of porcine (A), bovine (B), and human (C) OCTN2 and in vitro-translated mouse PPARα/RXRα. DIG-labeled specific and non-specific probes were used as positive and negative controls, respectively. Arrows indicate DNA/protein complexes and free DNA probes.
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Figure 4: Binding ofin vitrotranslated mouse PPARα/RXRα to the PPRE in the intron 1 of porcine, bovine and human OCTN2 gene. EMSA was performed using DIG-labeled oligonucleotides corresponding to the wild-type and the mutated PPRE of porcine (A), bovine (B), and human (C) OCTN2 and in vitro-translated mouse PPARα/RXRα. DIG-labeled specific and non-specific probes were used as positive and negative controls, respectively. Arrows indicate DNA/protein complexes and free DNA probes.

Mentions: To finally confirm that this PPRE is functional, we studied in vitro-binding of the PPARα/RXRα heterodimer to this PPRE using in vitro-translated PPARα/RXRα and labeled double-stranded oligonucleotides containing this PPRE sequence in gel shift assays (EMSA). As shown in Figure 4A-C, in the presence of PPARα/RXRα proteins, a band appeared representing a complex between the labeled oligonucleotide and the PPARα/RXRα heterodimer (lane 4, Figure 4A-C). On the contrary, no band for the DNA-PPARα/RXRα complex was observed when an oligonucleotide with a mutation in the putative PPRE was used (lane 5, Figure 4A-C). These observations indicated that the PPARα/RXRα heterodimer binds specifically to the identified PPRE in the intron 1 of porcine, bovine, and human OCTN2 gene, and this PPRE contributes to PPARα-dependent regulation of the OCTN2 gene in these species.


Transcriptional regulation of the human, porcine and bovine OCTN2 gene by PPARα via a conserved PPRE located in intron 1.

Luo H, Zhang Y, Guo H, Zhang L, Li X, Ringseis R, Wen G, Hui D, Liang A, Eder K, He D - BMC Genet. (2014)

Binding ofin vitrotranslated mouse PPARα/RXRα to the PPRE in the intron 1 of porcine, bovine and human OCTN2 gene. EMSA was performed using DIG-labeled oligonucleotides corresponding to the wild-type and the mutated PPRE of porcine (A), bovine (B), and human (C) OCTN2 and in vitro-translated mouse PPARα/RXRα. DIG-labeled specific and non-specific probes were used as positive and negative controls, respectively. Arrows indicate DNA/protein complexes and free DNA probes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4363911&req=5

Figure 4: Binding ofin vitrotranslated mouse PPARα/RXRα to the PPRE in the intron 1 of porcine, bovine and human OCTN2 gene. EMSA was performed using DIG-labeled oligonucleotides corresponding to the wild-type and the mutated PPRE of porcine (A), bovine (B), and human (C) OCTN2 and in vitro-translated mouse PPARα/RXRα. DIG-labeled specific and non-specific probes were used as positive and negative controls, respectively. Arrows indicate DNA/protein complexes and free DNA probes.
Mentions: To finally confirm that this PPRE is functional, we studied in vitro-binding of the PPARα/RXRα heterodimer to this PPRE using in vitro-translated PPARα/RXRα and labeled double-stranded oligonucleotides containing this PPRE sequence in gel shift assays (EMSA). As shown in Figure 4A-C, in the presence of PPARα/RXRα proteins, a band appeared representing a complex between the labeled oligonucleotide and the PPARα/RXRα heterodimer (lane 4, Figure 4A-C). On the contrary, no band for the DNA-PPARα/RXRα complex was observed when an oligonucleotide with a mutation in the putative PPRE was used (lane 5, Figure 4A-C). These observations indicated that the PPARα/RXRα heterodimer binds specifically to the identified PPRE in the intron 1 of porcine, bovine, and human OCTN2 gene, and this PPRE contributes to PPARα-dependent regulation of the OCTN2 gene in these species.

Bottom Line: Using positional cloning and reporter gene assays we identified a functional PPRE, each in the intron 1 of the porcine, bovine, and human OCTN2 gene.Gel shift assay confirmed binding of PPARα to this PPRE in the porcine, bovine, and the human OCTN2 gene.This suggests that regulation of genes involved in carnitine uptake by PPARα is highly conserved across species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Animal Husbandry and Veterinary Medicine, Shanxi Provincial Academy of Agricultural Sciences, Taiyuan, 030031, P. R. China. lhd2638@126.com.

ABSTRACT

Background: The novel organic cation transporter 2 (OCTN2) is the physiologically most important carnitine transporter in tissues and is responsible for carnitine absorption in the intestine, carnitine reabsorption in the kidney and distribution of carnitine between tissues. Genetic studies clearly demonstrated that the mouse OCTN2 gene is directly regulated by peroxisome proliferator-activated receptor α (PPARα). Despite its well conserved role as an important regulator of lipid catabolism in general, the specific genes under control of PPARα within each lipid metabolic pathway were shown to differ between species and it is currently unknown whether the OCTN2 gene is also a PPARα target gene in pig, cattle, and human. In the present study we examined the hypothesis that the porcine, bovine, and human OCTN2 gene are also PPARα target genes.

Results: Using positional cloning and reporter gene assays we identified a functional PPRE, each in the intron 1 of the porcine, bovine, and human OCTN2 gene. Gel shift assay confirmed binding of PPARα to this PPRE in the porcine, bovine, and the human OCTN2 gene.

Conclusions: The results of the present study show that the porcine, bovine, and human OCTN2 gene, like the mouse OCTN2 gene, is directly regulated by PPARα. This suggests that regulation of genes involved in carnitine uptake by PPARα is highly conserved across species.

Show MeSH