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

Breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) half transporter encoded by theAbcg2gene, 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 chickenAbcg2gene through cloning and characterization of its promoter region. Results showed that theAbcg2gene 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 ofAbcg2. 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 theAbcg2gene promoter. Interaction sites with theAbcg2gene 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 chickenAbcg2gene expression and the results may have far-reaching significance regarding the usage and development of veterinary drugs.Keywords:chickenAbcg2;promoter;regulators;LMH;primary hepatocytes