Identification of Functional Transcriptional Binding Sites within Chicken Abcg2 Gene Promoter and Screening Its Regulators.

Yujuan Zhang,Liping Wang,Jinhu Huang,Xiangxiu Li,Ci Fang

doi:10.3390/genes11020186

Yujuan Zhang, Liping Wang + Show 3 more

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https://doi.org/10.3390/genes11020186

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Abstract

Breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) half transporter encoded by the Abcg2 gene, is reported to influence the pharmacokinetics of substrate drugs during clinical therapy. The aim of this study was to clarify the mechanisms that regulate the transcription of the chicken Abcg2 gene through cloning and characterization of its promoter region. Results showed that the Abcg2 gene is transcribed by a TATA-less promoter with several putative Sp1 sites upstream from two putative CpG islands. A luciferase reporter assay conducted both in chicken leghorn male hepatoma (LMH) cells and chicken primary hepatocytes mapped a basal promoter to nucleotides −110 to +30, which is responsible for the constitutive expression of Abcg2. The 5′-region upstream of the basal promoter was characterized by both positive and negative regulatory domains. Further, using the cell-based reporter gene assay combined with RT-PCR and drug accumulation analysis, we found that four xenobiotics, daidzein, clotrimazole, ivermectin, and lipopolysaccharide (LPS), influence the expression and function of BCRP through significant regulation of the Abcg2 gene promoter. Interaction sites with the Abcg2 gene promoter of these four selected regulators were clarified by progressive deletions and mutation assays. This study shed some light on the regulatory mechanisms involved in chicken Abcg2 gene expression and the results may have far-reaching significance regarding the usage and development of veterinary drugs.

Highlights

It is currently recognized that the impact of drug transporters on clinically relevant drug disposition and drug–drug interactions (DDIs) is as significant as that of drug-metabolizing enzymes [1,2]
Breast cancer resistance protein (BCRP/ABCG2, encoded by the Abcg2 gene) is a known member of the ATP-binding cassette (ABC) transporter family that utilizes the hydrolysis of ATP to energize the efflux of a broad range of substrates, including commonly used antimicrobial agents licensed in veterinary medicine [3,4]
These results demonstrated that the putative CXR binding site was involved binding site was involved in the response of the Abcg2 gene promoter induced by daidzein

Summary

Introduction

It is currently recognized that the impact of drug transporters on clinically relevant drug disposition and drug–drug interactions (DDIs) is as significant as that of drug-metabolizing enzymes [1,2]. The FDA accepted BCRP as a key drug transporter involved in clinically relevant. DDIs, adverse drug reactions, and therapeutic failure of drugs due to its localization in organs that are important in drug disposition [5,6]. Unpredictable drug effects due to alterations in BCRP expression and activity have been frequently observed during clinical therapy [7,8,9]. It is of great clinical importance to elucidate the molecular mechanisms underlying the regulation of BCRP expression. Post-transcriptional and translational regulation are involved in BCRP regulation [10,11,12], it is well documented that the regulation of BCRP mainly occurs at the transcriptional level [13,14,15]

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