Cell Growth & Differentiation, Vol 7, Issue 9 1227-1237, Copyright © 1996 by American Association of Cancer Research
Localization of sequences that influence basal and cell type-specific activity of the murine mdr2 promoter
CP Yang, LS Kirschner, L Yu and SB Horwitz
Department of Molecular Pharmacology and Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
The mdr2 gene is highly expressed in liver and is involved in the
translocation of phospholipid. To study the regulation of mdr2 expression,
the promoter of the mdr2 gene has been isolated from a murine
vinblastine-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 transient
transfection assays were done in a variety of cell lines. The
promoter-luciferase construct displayed a 20- to 120-fold increase in
activity compared to the promoterless vector. 5' and 3' deletion analysis
using transient transfections revealed two major regulatory regions in the
promoter, one located upstream and one situated downstream of the
transcription start sites. The upstream region may be involved in basal
expression and the downstream sequence may be involved in cell
type-specific expression of the mdr2 gene. Gel mobility shift and DNA
footprinting assays have identified a 29-bp sequence (-78 to -50) to which
nuclear protein binds. Methylation interference analysis using this
fragment has further determined that CTGGCAGCTCGCCC, within the 29-mer,
contains the core sequence with which nuclear protein directly interacts.
Mutation of the core sequence reduced basal promoter activity, indicating
that it is involved in the basal expression of the mdr2 gene. Mutagenesis
studies also suggested that the upstream and downstream sequences act
independently in regulation of cell type-specific mdr2 expression.