Abstract
P-Glycoprotein (P-gp, Abcb1) plays a crucial role in drug disposition and functions by hydrolyzing ATP. However, little is known about the regulatory elements governing the transcription of the porcine Abcb1 gene. In this study, the transcription start site of the pig Abcb1 gene was identified by 5′-RACE. A 1.9-kb fragment of the 5′-flanking region of the Abcb1 gene was cloned from pig genomic DNA and sequenced. The region critical for its promoter activity was investigated via progressive deletions. Further, using mutation assays, two proximal Sp1 binding sites within the 5′-flanking region of Abcb1 were proven to be important cis-regulatory elements involved in regulating the constitutive expression of porcine Abcb1. RNA interference experiments showed that Sp1 regulated the expression of the porcine P-gp at both mRNA and protein levels. Hence, the current work provides valuable information on the regulatory mechanisms of pig Abcb1.
Highlights
ABC transporters are a large superfamily of transmembrane proteins and are able to transport a broad range of substrates with preference for hydrophobic or cationic compounds by hydrolyzing ATP (Fletcher et al, 2010)
By analyzing the promoter of pig ATP-binding cassette sub-family B member 1 (Abcb1) gene, we found that a GC rich region was located in the promoter, which was highly conserved in ABC transporter genes (Cornwell and Smith, 1993)
We found that the 5 -flanking region from −195 to +25 bp of porcine Abcb1 gene significantly influenced the promoter activity (Figure 3), suggesting that the core promoter is located in this region and the regulatory elements of this region may affect the promoter activity of porcine Abcb1
Summary
ABC transporters are a large superfamily of transmembrane proteins and are able to transport a broad range of substrates with preference for hydrophobic or cationic compounds by hydrolyzing ATP (Fletcher et al, 2010). Based on the important roles of the P-gp in swine drug disposition (Hsiu et al, 2002; Wang et al, 2004; Persson et al, 2008; Guo T. et al, 2016) and limited knowledge of the transcriptional regulatory mechanisms of the porcine Abcb gene, we characterized the 5 -flanking region of the porcine Abcb gene, and identified the core promoter region and cis-acting elements involved in the regulation of Abcb expression. Our results indicate that the transcription factor Sp1 can bind to the proximal promoter and is required to regulate the expression of porcine Abcb gene. Genomic DNA, used for amplifying the 5 -flanking sequence of the pig Abcb gene, was extracted from adult porcine jejunum using the Universal Genomic DNA Extraction Kit Ver. 5.0 (TaKaRa, Otsu, Japan). Amplicons were cloned using PMD18-T vector (TaKaRa, Otsu, Japan) and sequenced in both directions
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