Dataset Information

Cloning and Transcriptional Activity Analysis of the Porcine Abcb1 Gene Promoter: Transcription Factor Sp1 Regulates the Expression of Porcine Abcb1.

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.

SUBMITTER: Guo T

PROVIDER: S-EPMC5945876 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Cloning and Transcriptional Activity Analysis of the Porcine Abcb1 Gene Promoter: Transcription Factor Sp1 Regulates the Expression of Porcine Abcb1.

Guo Tingting T Huang Jinhu J Huan Changchao C He Fang F Zhang Yujuan Y Bhutto Zohaib A ZA Wang Liping L

Frontiers in pharmacology 20180504

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 ...[more]

PMID: 29780320

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Project description:Pigs are commonly used as an animal model to evaluate the toxic effects of exogenous compounds. Cytochrome P450 1A1 (CYP1A1) metabolizes numerous exogenous compounds and is abundantly expressed in the liver, kidneys, and intestines. The high amino acid similarity between human and porcine CYP1A1 indicates that they probably have the same metabolic characteristics. Therefore, understanding the regulatory mechanism of CYP1A1 expression in pigs is particularly important for predicting the toxicology and metabolic kinetics of exogenous chemicals. Currently, the transcriptional regulation of porcine CYP1A1 has rarely been studied, especially regarding basal transcription. In this study, we first confirmed that the key regulatory elements of porcine CYP1A1 basal transactivation are in the proximal promoter region using promoter truncation analysis via a dual luciferase assay in a porcine kidney cell line LLC-PK1. Two overlapping cis-elements, the xenobiotic response element (XRE) and GC box, in this proximal region potentially play key roles in the basal transactivation of porcine CYP1A1. Furthermore, using electrophoretic mobility shift assay and chromatin immunoprecipitation, the GC box binding protein Sp1 was confirmed to bind to the proximal promoter of porcine CYP1A1, instead of AhR, the XRE binding protein. In LLC-PK1 cells, by knocking down either Sp1 or AhR, the expression of porcine CYP1A1 at the mRNA level and protein level was significantly downregulated, suggesting both proteins are important for porcine CYP1A1 expression. However, promoter activity analysis in LLC-PK1 cells treated with an AhR agonist and antagonist confirmed that AhR does not participate in the basal regulation of porcine CYP1A1 at the proximal promoter. In conclusion, our study revealed that the proximal promoter is the key regulatory region for porcine CYP1A1 basal expression. Although AhR plays an important role in the transactivation of porcine CYP1A1 expression, the key determinant transcription factor for its basal transactivation is Sp1 at the proximal promoter of porcine CYP1A1.

Dataset Information

Cloning and Transcriptional Activity Analysis of the Porcine Abcb1 Gene Promoter: Transcription Factor Sp1 Regulates the Expression of Porcine Abcb1.

Publications

Cloning and Transcriptional Activity Analysis of the Porcine Abcb1 Gene Promoter: Transcription Factor Sp1 Regulates the Expression of Porcine Abcb1.

Similar Datasets

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