Abstract
BackgroundThe BRCA1 gene plays an important role in the maintenance of genomic stability. BRCA1 inactivation contributes to breast cancer tumorigenesis. An increasing number of transcription factors have been shown to regulate BRCA1 expression. c-Myc can act as a transcriptional activator, regulating up to 15% of all genes in the human genome and results from a high throughput screen suggest that BRCA1 is one of its targets. In this report, we used cultured breast cancer cells to examine the mechanisms of transcriptional activation of BRCA1 by c-Myc.Methodsc-Myc was depleted using c-Myc-specific siRNAs in cultured breast cancer cells. BRCA1 mRNA expression and BRCA1 protein expression were determined by quantitative RT-PCR and western blot, respectively and BRCA1 promoter activities were examined under these conditions. DNA sequence analysis was conducted to search for high similarity to E boxes in the BRCA1 promoter region. The association of c-Myc with the BRCA1 promoter in vivo was tested by a chromatin immunoprecipitation assay. We investigated the function of the c-Myc binding site in the BRCA1 promoter region by a promoter assay with nucleotide substitutions in the putative E boxes. BRCA1-dependent DNA repair activities were measured by a GFP-reporter assay.ResultsDepletion 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. In the distal BRCA1 promoter, DNA sequence analysis revealed two tandem clusters with high similarity, and each cluster contained a possible c-Myc binding site. c-Myc bound to these regions in vivo. Nucleotide substitutions in the c-Myc binding sites in these regions abrogated c-Myc-dependent promoter activation. Furthermore, breast cancer cells with reduced BRCA1 expression due to depletion of c-Myc exhibited impaired DNA repair activity.ConclusionsThe distal BRCA1 promoter region is associated with c-Myc and contributes to BRCA1 gene activation.
Highlights
The BRCA1 gene plays an important role in the maintenance of genomic stability
We show that depletion of cMyc is correlated with a reduction in BRCA1 mRNA and BRCA1 protein levels and decreased BRCA1 promoter activities were observed in the cells following depletion of c-Myc
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 Small interfering RNA (siRNA) treated cells (Figure 1B)
Summary
The BRCA1 gene plays an important role in the maintenance of genomic stability. BRCA1 inactivation contributes to breast cancer tumorigenesis. C-Myc can act as a transcriptional activator, regulating up to 15% of all genes in the human genome and results from a high throughput screen suggest that BRCA1 is one of its targets. We used cultured breast cancer cells to examine the mechanisms of transcriptional activation of BRCA1 by c-Myc. The human breast cancer susceptibility gene 1 product, BRCA1 is involved in important cellular processes, including DNA repair, and loss of BRCA1 can result in genomic instability. The E2F family of transcription factors binds to two regions, -41 to -31 and -21 to -11, and activates or represses BRCA1 expression depending on the cofactors recruited [18,19], BRCA1 itself has been shown to be one of the co-factors [20]. A relatively long segment in a 5 kb region in BRCA1 intron 2 that is highly conserved in multiple species contains a CNS-1 (Conserved Nucleotide Site-1) and CNS-2, which appear to act as repression and activation elements, respectively [24]
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