Shimizu M, Nomura Y, Suzuki H, Ichikawa E, Takeuchi A, Suzuki M, Nakamura T, Nakajima T, Oda K
Department of Biological Science and Technology, Science University of Tokyo, Japan.
Exp Cell Res. 1998 Feb 25;239(1):93-103. doi: 10.1006/excr.1997.3884.
Cyclin A plays an essential role in the G1 to S phase transition in the cell cycle. The expression of cyclin A is restrained during G0 and G1, but steeply induced at the G1/S boundary. Analysis of the rat cyclin A promoter elements with the 5' sequential deletion derivatives of the promoter fused to the luciferase cDNA indicated that the ATF/CRE motif primarily determines the inducibility at G1/S. Gel shift analysis of the complex formed at the ATF/CRE site indicated that the complex was not formed with the G0/G1 cell extract, but maximally formed with the late-G1 cell extract. The complex was supershifted by anti-JunD antibody, and Western blot analysis of the immune complexes prepared with anti-JunD antibody revealed the presence of ATF2, suggesting heterodimerization of JunD with ATF2. The cyclin A promoter in a reporter plasmid was activated by nearly 10-fold in quiescent rat 3Y1 cells by cotransfection with the expression of plasmids encoding ATF2 and Jun family members. In contrast, cotransfection with the ATF4 expression plasmid suppressed the promoter activation mediated by ATF2 and Jun family members. The expression of Jun family members during G1 to S progression was induced biphasically in early and late G1 and the level of JunD increased markedly at the G1/S, while that of ATF family members was gradually increased along with the G1 to S progression. These results indicate that the cyclin A promoter activity is regulated, at least in part, by relative amounts of the ATF and Jun family members.
细胞周期蛋白A在细胞周期的G1期到S期转换过程中发挥着至关重要的作用。细胞周期蛋白A的表达在G0期和G1期受到抑制，但在G1/S边界处急剧诱导。用与荧光素酶cDNA融合的启动子的5'序列缺失衍生物对大鼠细胞周期蛋白A启动子元件进行分析表明，ATF/CRE基序主要决定了G1/S期的诱导性。对在ATF/CRE位点形成的复合物进行凝胶迁移分析表明，该复合物在G0/G1期细胞提取物中未形成，但在G1晚期细胞提取物中大量形成。该复合物被抗JunD抗体超迁移，用抗JunD抗体制备的免疫复合物的蛋白质免疫印迹分析显示存在ATF2，提示JunD与ATF2发生异源二聚化。通过与编码ATF2和Jun家族成员的表达质粒共转染，报告质粒中的细胞周期蛋白A启动子在静止的大鼠3Y1细胞中被激活了近10倍。相反，与ATF4表达质粒共转染抑制了由ATF2和Jun家族成员介导的启动子激活。在G1期到S期进展过程中，Jun家族成员的表达在G1早期和晚期呈双相诱导，JunD的水平在G1/S期显著增加，而ATF家族成员的水平则随着G1期到S期的进展而逐渐增加。这些结果表明，细胞周期蛋白A启动子活性至少部分受ATF和Jun家族成员相对含量的调节。
