Abstract
BackgroundBRCA2 gene expression is tightly regulated during the cell cycle in human breast cells. The expression of BRCA2 gene is silenced at the G0/G1 phase of cell growth and is de-silenced at the S/G2 phase. While studying the activity of BRCA2 gene promoter in breast cancer cells, we discovered that this promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter at the G0/G1 phase of the cell. Standard techniques like cell synchronization by serum starvation, flow cytometry, N-terminal or C-terminal FLAG epitope-tagged protein expression, immunofluorescence confocal microscopy, dual luciferase assay for promoter evaluation, and chromatin immunoprecipitation assay were employed during this study.ResultsHuman BRCA2 gene promoter is active in both the forward and the reverse orientations. This promoter is 8-20 fold more active in the reverse orientation than in the forward orientation when the cells are in the non-dividing stage (G0/G1). When the cells are in the dividing state (S/G2), the forward activity of the promoter is 5-8 folds higher than the reverse activity. The reverse activity transcribes the ZAR2 mRNA with 966 nt coding sequence which codes for a 321 amino acid protein. ZAR2 has two C4 type zinc fingers at the carboxyl terminus. In the G0/G1 growth phase ZAR2 is predominantly located inside the nucleus of the breast cells, binds to the BRCA2 promoter and inhibits the expression of BRCA2. In the dividing cells, ZAR2 is trapped in the cytoplasm.ConclusionsBRCA2 gene promoter has bi-directional activity, expressing BRCA2 and a novel C4-type zinc finger containing transcription factor ZAR2. Subcellular location of ZAR2 and its expression from the reverse promoter of the BRCA2 gene are stringently regulated in a cell cycle dependent manner. ZAR2 binds to BRCA2/ZAR2 bi-directional promoter in vivo and is responsible, at least in part, for the silencing of BRCA2 gene expression in the G0/G1 phase in human breast cells.
Highlights
BRCA2 gene expression is tightly regulated during the cell cycle in human breast cells
Our studies suggest that negative regulation of BRCA2 gene expression by the ZAR1-like protein (ZAR2) at the G0/G1 phase of human breast cell growth may provide an additional mechanism of cell cycle-dependent regulation of its expression in both SLUG-positive and SLUG-negative cells
We developed a dual reporter construct in which the firefly luciferase (Fluc) gene ORF is transcribed by the reverse activity of the cloned promoter and the forward activity of the promoter will transcribe Renilla luciferase (Rluc) gene ORF (Fig 1B) from a single plasmid DNA
Summary
BRCA2 gene expression is tightly regulated during the cell cycle in human breast cells. While studying the activity of BRCA2 gene promoter in breast cancer cells, we discovered that this promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter at the G0/G1 phase of the cell. The tumor suppressor protein BRCA2 is implicated in the regulated growth and proliferation of human breast [1,2,3,4], prostate [5,6], ovarian [7,8], esophageal [9], and pancreatic [10,11] cells. The notion could be that unique cellular mechanisms are triggered in the breast cancer cells to stimulate BRCA2 gene expression as a temporary measure to regulate the growth of the breast cancer cells. One potential mechanism of BRCA2 involvement in breast cancer progression may be through deregulation of the BRCA2 gene expression
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