LOCALIZATION OF SEQUENCES THAT INFLUENCE BASAL AND CELL-TYPE-SPECIFICACTIVITY OF THE MURINE MDR2 PROMOTER

The mdr2 gene is highly expressed in liver and is involved in the tran slocation of phospholipid. To study the regulation of mdr2 expression, the promoter of the mdr2 gene has been isolated from a murine vinblas tine-resistant cell line, J7.V2-1, and characterized, The 5' flanking region of this gene is GC-rich, has multiple transcription initiation sites as mapped by primer extension, and does not contain either TATA or CCAAT boxes, To test promoter activity, a 1.9-kb (-1867 to +37) DNA fragment was cloned in front of the luciferase reporter gene and tran sient transfection assays were done in a variety of cell lines, The pr omoter-luciferase construct displayed a 20- to 120-fold increase in ac tivity compared to the promoterless vector. 5' and 3' deletion analysi s using transient transfections revealed two major regulatory regions in the promoter, one located upstream and one situated downstream of t he transcription start sites, The upstream region may be involved in b asal expression and the downstream sequence may be involved in cell ty pe-specific expression of the mdr2 gene, Gel mobility shift and DNA fo otprinting assays have identified a 29-bp sequence (-78 to -50) to whi ch nuclear protein binds, Methylation interference analysis using this fragment has further determined that CTGGCAGCTCGCCC, within the 29-me r, contains the core sequence with which nuclear protein directly inte racts, Mutation of the core sequence reduced basal promoter activity, indicating that if is involved in the basal expression of the mdr2 gen e, Mutagenesis studies also suggested that the upstream and downstream sequences act independently in regulation of cell type-specific mdr2 expression.