Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal Promoter
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 ind...
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| Language: | English |
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Frontiers Media S.A.
2018-08-01
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| Series: | Frontiers in Pharmacology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fphar.2018.00927/full |
| _version_ | 1818541475598696448 |
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| author | Xuan Xie Xuan Xie Jun Jiang Jun Jiang Wenchu Ye Wenchu Ye Ruohong Chen Ruohong Chen Yiqun Deng Yiqun Deng Jikai Wen Jikai Wen |
| author_facet | Xuan Xie Xuan Xie Jun Jiang Jun Jiang Wenchu Ye Wenchu Ye Ruohong Chen Ruohong Chen Yiqun Deng Yiqun Deng Jikai Wen Jikai Wen |
| author_sort | Xuan Xie |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-11T22:09:46Z |
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| institution | Directory Open Access Journal |
| issn | 1663-9812 |
| language | English |
| last_indexed | 2024-12-11T22:09:46Z |
| publishDate | 2018-08-01 |
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| spelling | doaj.art-037f2ea13d0e48f899baab70fdf4294a2022-12-22T00:48:50ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122018-08-01910.3389/fphar.2018.00927400686Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal PromoterXuan Xie0Xuan Xie1Jun Jiang2Jun Jiang3Wenchu Ye4Wenchu Ye5Ruohong Chen6Ruohong Chen7Yiqun Deng8Yiqun Deng9Jikai Wen10Jikai Wen11Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, ChinaKey Laboratory of Zoonosis of Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, ChinaKey Laboratory of Zoonosis of Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, ChinaKey Laboratory of Zoonosis of Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, ChinaKey Laboratory of Zoonosis of Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, ChinaKey Laboratory of Zoonosis of Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, ChinaKey Laboratory of Zoonosis of Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, ChinaPigs 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.https://www.frontiersin.org/article/10.3389/fphar.2018.00927/fullporcineCYP1A1Sp1AhRbasal transactivation |
| spellingShingle | Xuan Xie Xuan Xie Jun Jiang Jun Jiang Wenchu Ye Wenchu Ye Ruohong Chen Ruohong Chen Yiqun Deng Yiqun Deng Jikai Wen Jikai Wen Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal Promoter Frontiers in Pharmacology porcine CYP1A1 Sp1 AhR basal transactivation |
| title | Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal Promoter |
| title_full | Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal Promoter |
| title_fullStr | Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal Promoter |
| title_full_unstemmed | Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal Promoter |
| title_short | Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal Promoter |
| title_sort | sp1 instead of ahr regulates the basal transcription of porcine cyp1a1 at the proximal promoter |
| topic | porcine CYP1A1 Sp1 AhR basal transactivation |
| url | https://www.frontiersin.org/article/10.3389/fphar.2018.00927/full |
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