Hu Z, Jin S, Scotto K W
Program in Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
J Biol Chem. 2000 Jan 28;275(4):2979-85. doi: 10.1074/jbc.275.4.2979.
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 a repressive 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.
多药耐药基因1（MDR1）启动子受多种内部和外部刺激的调控。我们之前已经表明，CCAAT盒结合蛋白NF-Y通过募集共激活因子P/CAF介导组蛋白去乙酰化酶抑制剂曲古抑菌素A和丁酸钠对MDR1的激活作用。我们现在将研究扩展至基因毒性应激对MDR1的激活作用。我们发现紫外线照射对MDR1启动子的激活同样依赖于CCAAT盒（-82至-73）以及一个近端GC元件（-56至-42）。用人KB-3-1细胞制备的核提取物进行凝胶迁移和超迁移分析，确定NF-Y为与CCAAT盒相互作用的转录因子，而Sp1是与GC元件结合的主要因子。消除这些因子中任何一个结合的突变会降低或消除紫外线照射引起的激活作用；此外，共表达显性负性NF-Y蛋白（NF-YA29）会降低紫外线激活的转录。有趣的是，转录因子YB-1也识别CCAAT基序，并且据报道可介导紫外线对MDR1启动子的诱导作用，尽管它确实能与单链寡核苷酸相互作用，但它在核提取物中无法与双链MDR1 CCAAT盒寡核苷酸相互作用。此外，消除紫外线照射对MDR1激活作用的突变对YB-1的结合没有影响，并且YB-1表达质粒的共转染对紫外线诱导的转录有抑制作用。综上所述，这些结果表明NF-Y和Sp1在基因毒性应激对MDR1基因的转录激活中均发挥作用，并且表明YB-1即使参与其中，也不足以介导这种激活作用。
