Transcriptional Activation of the MDR1 Gene by UV Irradiation
ROLE OF NF-Y AND Sp1*
- From the Program in Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Abstract
The MDR1 promoter is subject to control by various internal and external stimuli. We have previously shown that the CCAAT box-binding protein, NF-Y, mediates MDR1 activation by the histone deacetylase inhibitors, trichostatin A and sodium butyrate, through the recruitment of the co-activator, P/CAF. We have now extended our investigation to the activation of MDR1 by genotoxic stress. We show that activation of the MDR1 promoter by UV irradiation is also dependent on the CCAAT box (−82 to −73) as well as on a proximal GC element (−56 to −42). Gel shift and supershift analyses with nuclear extracts prepared from human KB-3-1 cells identified NF-Y as the transcription factor interacting with the CCAAT box, while Sp1 was the predominant factor binding to the GC element. Mutations that abrogated binding of either of these factors reduced or abolished activation by ultraviolet irradiation; moreover, co-expression of a dominant-negative NF-Y protein (NF-YA29) reduced UV-activated transcription. Interestingly, YB-1, a transcription factor that also recognizes the CCAAT motif and had been reported to mediate induction of the MDR1 promoter by ultraviolet light, was incapable of interacting with the double-stranded MDR1 CCAAT box oligonucleotide in nuclear extracts, although it did interact with a single-stranded oligonucleotide. Furthermore, a mutation that abolished activation of MDR1 by UV-irradiation had no effect on YB-1 binding and co-transfection of a YB-1 expression plasmid had arepressive effect on UV-inducible transcription. Taken together, these results indicate a role for both NF-Y and Sp1 in the transcriptional activation of the MDR1 gene by genotoxic stress, and indicate that YB-1, if involved, is not sufficient to mediate this activation.
Footnotes
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↵* This work was supported by National Cancer Institute Grants P30-CA-08748 (to Memorial Sloan-Kettering Cancer Center) and RO1-CA-57307 (to K. W. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Program in Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, New York 10021. Tel.: 212-639-8972; Fax: 212-639-2767; E-mail: k-scotto@ski.mskcc.org.
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↵2 B. Gorfajn, S. Jin, and K. W. Scotto, manuscript in preparation.
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↵3 S. Jin and K. W. Scotto, manuscript in preparation.
- Abbreviations:
- Pgp
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P-glycoprotein
- MDR
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multidrug resistance
- ds
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double-stranded
- DTT
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dithiothreitol
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- Received August 5, 1999.
- Revision received September 23, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
