c-Myc activates BRCA1 gene expression through distal promoter elements in breast cancer cells.
Bottom Line: Depletion of c-Myc was found to be correlated with reduced expression levels of BRCA1 mRNA and BRCA1 protein.Depletion of c-Myc decreased BRCA1 promoter activity, while ectopically expressed c-Myc increased BRCA1 promoter activity.The distal BRCA1 promoter region is associated with c-Myc and contributes to BRCA1 gene activation.
Affiliation: Center for Clinical Cancer Genetics and Global Health, Department of Medicine, University of Chicago, Chicago, IL, USA.Show MeSH
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Mentions: BRCA1 has been implicated as a c-Myc target in a high throughput study using human umbilical vein endothelial cells overexpressing c-Myc . Whether c-Myc can directly regulate BRCA1 expression at the transcriptional level is not clear. We successfully depleted c-Myc expression in the breast cancer cell lines MCF-7 and MDA-MB-231 through treatment with siRNA against the c-Myc gene. In both cell lines, transient transfection of c-Myc siRNA efficiently reduced c-Myc protein expression (Figure 1A). Correspondingly, BRCA1 protein levels were reduced upon depletion of c-Myc in both cell lines (Figure 1A). More importantly, BRCA1 mRNA levels in cells with depleted c-Myc protein were reduced to 24% in MCF-7 and 36% in MDA-MB-231 compared to the control siRNA treated cells (Figure 1B). To establish that BRCA1 expression was directly modulated by c-Myc through DNA regulatory elements in the BRCA1 promoter region, we constructed a BRCA1 promoter/luciferase reporter vector containing the BRCA1 promoter region -1714 to +42. BRCA1 promoter activity following depletion of c-Myc was significantly reduced compared to control in both cell lines (Figure 1C). These data suggest that reduced c-Myc might decrease BRCA1 expression in these two breast cancer cell lines through the BRCA1 promoter. To test whether this BRCA1 promoter region sufficiently responded to c-Myc activation, we monitored BRCA1 promoter activity in the presence of ectopically expressed functional c-Myc from transient transfections of pMYC-GFP into MCF-7 or MDA-MB-231 cells. We observed increasing expression of the Myc-GFP with increasing amounts of Myc-GFP expression vector used, while the endogenous c-Myc expression remained consistent (Figure 2A). In both cultured cell types, Myc-GFP protein levels produced at the maximal dose of the Myc-GFP expression vector were similar to the levels of endogenous c-Myc at the time when cells were used for luciferase assays (Figure 2A). In the presence of constitutively expressed Myc-GFP, BRCA1 promoter activity increased proportionally to increasing amounts of pMyc-GFP (Figure 2B). Taken together, these data indicated that c-Myc activates BRCA1 expression through the region of the BRCA1 promoter (-1714 to +42), which could contain c-Myc-mediated DNA regulatory elements.
Affiliation: Center for Clinical Cancer Genetics and Global Health, Department of Medicine, University of Chicago, Chicago, IL, USA.