کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1946672 | 1054263 | 2011 | 11 صفحه PDF | دانلود رایگان |
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 − 42 bps 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.
Research highlights
► Human intestinal BCRP expression is regulated by the alternative promoter E1C.
► Mouse intestinal Bcrp1 expression is regulated by the alternative promoter E1b.
► A p-CREB response element (CRE) was found in mouse E1b promoter but not in the other mouse alternative Bcrp1 promoters.
► p-CREB directly binds and activates transcription from Bcrp1 E1b promoter.
► cAMP plays a role in regulation of mouse small intestinal Bcrp1 transcription.
Journal: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms - Volume 1809, Issue 7, July 2011, Pages 295–305