CCAAT/enhancer-binding protein β (nuclear factor for interleukin 6) transactivates the human MDR1 gene by interaction with an inverted CCAAT box in human cancer cells

We investigated the mechanisms of MDR1 gene activation by CCAAT/enhancer binding protein beta (C/EBPbeta, or nuclear factor for interleukin 6) in human cancer cells. Transfection of the breast cancer cell line MCF-7 and its doxorubicin-selected variant MCF-7/ADR by either C/EBPbeta or C/EBPbeta-LIP (a dominant-negative form of C/EBPbeta) confirmed their roles in the activation or repression of the endogenous, chromosomally embedded MDR1 gene. Cotransfection experiments with promoter constructs revealed a C/EBPbeta interaction on the MDR1 promoter via the region within -128 to -75. Deletions within the putative AP-1 box (-123 to -111) increased MDR1 promoter activity when stimulated by C/EBPbeta, suggesting that the AP-1 site negatively regulates MDR1 activation by C/EBPbeta. Mutations within the inverted CCAAT box (Y box) (-82 to -73) abolished the C/EBPbeta-stimulated MDR1 promoter activity, indicating that the Y box is required for MDR1 activation by C/EBPbeta. Chromatin immunoprecipitation (ChIP) revealed that C/EBPbeta precipitates a transcription complex containing C/EBPbeta, the MDR1 promoter sequences (-250 to +54), and the hBrm protein. In conclusion, alteration of expression or function of C/EBPbeta plays an important role in MDR1 gene regulation. C/EBPbeta activates the endogenous MDR1 gene of MCF-7 cells, and this activation was associated with a novel C/EBPbeta interaction region within the proximal MDR1 promoter (-128 to -75). The mechanisms of MDR1 activation by C/EBPbeta include C/EBPbeta binding of the chromatin of the MDR1 gene and interactions of C/EBPbeta with the Y box and Y box-associated proteins.