DNA Elements Recognizing NF-Y and Sp1 Regulate the Human Multidrug-Resistance Gene Promoter
- 1Division of Molecular Genetics and Microbiology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709 (R.S., A.C.K.), and 2Lineberger Cancer Research Center and Department of Microbiology-Immunology Univ. of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 (G.M., J.T.)
Abstract
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.
Footnotes
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Send reprint requests to: Dr. Rebecca Sundseth, AndCare, P.O. Box 14566, Research Triangle Park, NC 27709.
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↵1 Current affiliation: BioChem Pharma, Inc., Laval, Quebec, Canada, H7V 4A7.
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↵2 A. Merritt and A. C. King, unpublished observations.
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This work was supported by Burroughs Wellcome Co. and Grants CA48185, CA37172, and AI27430 from the National Cancer Institute and National Institute of Allergy and Infectious Diseases (J.T.) and a predoctoral training grant from the National Institutes of Health (G.M.).
- Abbreviations:
- hMDR
- human multidrug resistance
- DMEM
- Dulbecco’s modified Eagle’s medium
- EGTA
- ethylene glycol bis(α-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- PCR
- polymerase chain reaction
- DTT
- dithiothreitol
- DOTAP
- N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate
- SV
- simian virus
- LU
- light units
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- Received January 30, 1996.
- Accepted February 18, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
