The Mouse Plasma Membrane Ca²⁺-ATPase Isoform2bw Gene Promoter: Cloning And Characteristics Analysis Of Transcriptional Regulation

Milk is the most calcium rich biological fluid, which requires mothers transport lots ofcalcium across mammary epithelia cells to milk everyday. Calcium homeostasis is necessaryfor all eukaryotic cells. Therefore, there must have a high capacity transport machinery totransport calcium against a concentration gradient from cells to milk. Atp2b2encodes theplasma membrane Ca2+-ATPase type2(PMCA2) expressed in cells. PMCA2wb is aspliceosome of PMCA2. PMCA2wb is responsible for the transportation60%calcium ionsinto the milk. PMCA2wb is ositively correlated with the severity of the breast cancer. Thosesuggest that precise regulation of PMCA2wb is very important in mammary land. Therefore,study of the structure and functions of PMCA2wb promoter can further reveal regulatorymechanisms of PMCA2wb on transcription level.This study cloned PMCA2wb gene promoter of mouse by PCR, and conducted sequenceanalysis upon the promoter. Fragments of the promoter of different lengths were obtained bydeletion and sub-cloned into expression vectors with a luciferase reporter gene. The vectorswere transfected into mammary epithelial cells and MCF-7cells, and their activities ofexpression were measured using dual-luciferase reporter assay system. The core region of thepromoter with basal transcriptional activity was determined with dual-luciferase reporterassays. The effect of transcriptional factor binding sites on promoter activity was detected viaoverlap extension PCR. The results are as follows:(1) A sequence of2576bp of5’flanking sequence of PMCA2wb gene of mouse wassuccessfully cloned, which include2373bp upstream transcription start site (+1), partial exonI203bp. There is no classic TATA box on this sequence.(2) Fragments of the promoter of different length obtained by deletion mutantion wereconnected with a luciferase reporter plasmide to form a recombinant plasmid. Test data ofpromoter activity was analysed after MECs was transfected by recombinant plasmid. Theresult show that the core region of PMCA2wb gene ranged from-287bp to+8bp. (3) The results of site-directed mutagenesis showed that mutation in CREB (-268bp)、USF (-229bp) and Sp1(-200bp) binding sites significantly could reduce the promoter activity by33%(P <0.01),37%(P <0.01) and22%(0.01<P<0.05). Results indicated that CREB, USFand Sp1maybe regulate expression of PMCA2wb gene on transcriptional level throughbinding onto the promoter of PMCA2wb.In conclusion, this study cloned the5’ end sequence of mouse PMCA2wb gene. Theresult of deletion analysis revealed that the core region of PMCA2wb gene promoter is from-287bp to+8bp. The result of site directed mutagenesis showed that CREB, USF and Sp1maybe involve in regulating the expression of PMCA2wb gene on transcriptional level.