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Division of Molecular Genetics and Microbiology, Wellcome Research
Laboratories, Research Triangle Park, North Carolina 27709 (R.S.,
A.C.K.), and
Lineberger Cancer Research Center and Department of
Microbiology-Immunology Univ. of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599 (G.M., J.T.)
Regulation of the human multidrug resistance gene
(hMDR1) was studied by mapping DNA elements in the
proximal promoter necessary for efficient transcription. Transient
transfection analysis in tumor cell lines (HCT116, HepG2, and Saos2) of
promoter deletions identified several regulatory domains. These cell
lines expressed hMDR1 mRNA. Removal of an element between +25 and +158
reduced promoter activity by 2-3-fold, whereas deletion of sequences
from ~
5000 to 138 base pairs gave a ~2-fold increase. The
activity of the hMDR1 promoter (137 to +25) was comparable
in activity to the SV40 early promoter and enhancer combination.
Deletion of the hMDR1 promoter between 86 and 44 reduced
activity by 5-10-fold, identifying an important regulatory region.
This minimal region (88 to 37) activated transcription when
inserted upstream of a synthetic promoter, suggesting that it acts
independently of other regulatory sequences. Two DNA elements within 85 base pairs of the transcriptional start site were required to confer efficient gene expression. A double-point mutation in the Y box (inverted CCAAT box) between 70 and 80 reduced activity of the promoter by 5-10-fold, and a single-point mutation at 52 within a
GC-rich element reduced activity by 3-fold. Thus, both the Y-box and GC
elements must each remain intact for optimal promoter activity. DNA-binding analyses suggest that the transcription factor NF-Y, but
not YB-1 or c/EBP, is most likely responsible for controlling the
activity of the Y-box element in these tumor cell lines. DNA-binding analyses also suggest that Sp1, alone or in combination with other nuclear factors, likely controls the activity of the GC element.
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