Division of Biomedical Sciences, Meharry Medical College, Nashville, TN 37208, USA.
Mol Cancer. 2010 Mar 4;9:50. doi: 10.1186/1476-4598-9-50.
BRCA2 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.
Human 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.
BRCA2 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.
BRCA2 基因在人类乳腺细胞的细胞周期中受到严格调控。BRCA2 基因的表达在细胞生长的 G0/G1 期被沉默，并在 S/G2 期被去沉默。在研究乳腺癌细胞中 BRCA2 基因启动子的活性时，我们发现该启动子具有双向活性，反向活性的产物（一种 ZAR1 样蛋白，我们命名为 ZAR2）在细胞的 G0/G1 期沉默正向启动子。在这项研究中，使用了细胞同步化（如血清饥饿、流式细胞术、N 端或 C 端 FLAG 表位标记蛋白表达、免疫荧光共聚焦显微镜、用于启动子评估的双荧光素酶测定法和染色质免疫沉淀测定法等标准技术。
人 BRCA2 基因启动子在正向和反向两个方向上均具有活性。当细胞处于非分裂状态（G0/G1）时，该启动子在反向方向上的活性比正向方向上高 8-20 倍。当细胞处于分裂状态（S/G2）时，启动子的正向活性比反向活性高 5-8 倍。反向活性转录出具有 966 个核苷酸编码序列的 ZAR2 mRNA，编码一个 321 个氨基酸的蛋白质。ZAR2 在羧基末端具有两个 C4 型锌指。在 G0/G1 生长阶段，ZAR2 主要位于乳腺细胞的核内，与 BRCA2 启动子结合并抑制 BRCA2 的表达。在分裂细胞中，ZAR2 被捕获在细胞质中。
BRCA2 基因启动子具有双向活性，表达 BRCA2 和一种新型的 C4 型锌指含转录因子 ZAR2。ZAR2 的亚细胞定位及其从 BRCA2 基因的反向启动子表达在细胞周期依赖性方式中受到严格调控。ZAR2 在体内与 BRCA2/ZAR2 双向启动子结合，至少部分负责在人乳腺细胞的 G0/G1 期沉默 BRCA2 基因的表达。
