title: Identification and characterization of the major alternative promoter regulating Bcrp1/Abcg2 expression in the mouse intestine.
abstract: Mouse models are often used to predict drug absorption in humans. Mouse Bcrp1 protein exhibits sequence and functional homology with human BCRP protein. Additionally, BCRP/Bcrp1 expression is regulated by alternative promoter usage in humans and mice; however, the precise intestine-specific alternative promoter utilized in either species is yet to be determined. Therefore we sought to identify and characterize the mouse intestinal Bcrp1 promoter. Using real-time quantitative RT-PCR and 5' RACE PCR we first established the predominance of a single Bcrp1 first exon (E1b) in the Bcrp1 mRNA isolated throughout the mouse intestine. Simultaneously using 5' RACE PCR we identified E1C as the predominant BCRP 5' UTR expressed in the human intestine. Next we established functional activity for the murine promoter upstream of E1b using reporter assays. Subsequently using deletion-construct analysis we found the core promoter region to span -231 to -42bps from the transcriptional start site of E1b. We then predicted a cAMP response element (CRE) as a transcription factor binding site unique only to the E1b promoter region, using in silico methods. We finally established functional interaction of phospho-CREB (p-CREB) protein with the CRE on the E1b promoter using both functional assays and chromatin immunoprecipitation assays. In conclusion, mouse intestinal Bcrp1 expression is regulated by a single alternative promoter upstream of E1b, the predominant Bcrp1 mRNA isoform expressed in the mouse intestine. Furthermore, Bcrp1 E1b mRNA expression is regulated by binding of p-CREB to its cis site on the mouse E1b promoter region.
authors: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.
authors: Karthika
authors: K
authors: Natarajan
authors: Yi
authors: Y
authors: Xie
authors: Takeo
authors: T
authors: Nakanishi
authors: William T
authors: WT
authors: Beck
authors: Kenneth S
authors: KS
authors: Bauer
authors: Douglas D
authors: DD
authors: Ross
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D051379:Mice; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D009928:Organ Specificity / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D020218:Response Elements / Q000502:physiology*; D013045:Species Specificity
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; C501412:Abcg2 protein, mouse; C115163:CREB1 protein, human; C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D009363:Neoplasm Proteins; D012333:RNA, Messenger