Dou Q P, Zhao S, Levin A H, Wang J, Helin K, Pardee A B
Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, Massachusetts.
J Biol Chem. 1994 Jan 14;269(2):1306-13.
By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes. (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide interchanged these complexes with similar kinetics. (v) When MT2-shifted E2F.G0/G1, E2F.S, and free E2F were eluted and analyzed by Western blot assay using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositions of these MT2-bound complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene.
通过进行DNA酶I足迹分析，我们已鉴定出位于小鼠胸苷激酶（TK）上游启动子上的三个不同的蛋白质结合序列（MT1、MT2和MT3）（Dou，Q.-P.，Fridovich-Keil，J.L.，和Pardee，A.B.（1991年）《美国国家科学院院刊》88，1157 - 1161）。在此我们报告，如下列证据所示，MT2包含一个E2F样结合位点（GTTCGCGGGCAAA）。（i）MT2特异性结合亲和纯化的融合人E2F蛋白。（ii）MT2和一个真实的E2F位点（TTTCGCGCGCTTT）都特异性结合相似或相同的核蛋白复合物。（iii）这两种DNA - 蛋白质复合物的形成均依赖细胞周期：一个G0/G1期特异性复合物（E2F.G0/G1）被一个S期特异性复合物（E2F.S）取代，而“游离”的E2F在G1/S转变后增加。（iv）用环己酰亚胺脉冲抑制蛋白质合成以相似的动力学交换了这些复合物。（v）当MT2迁移的E2F.G0/G1、E2F.S和游离E2F被洗脱并用针对人E2F - 1的特异性抗血清通过蛋白质印迹分析进行分析时，从所有三种复合物中均观察到两种形式的小鼠E2F（62和66 kDa）。还对这些与MT2结合的复合物的组成进行了研究。使用特异性抗体的研究表明，视网膜母细胞瘤样蛋白p107存在于E2F - G0/G1和E2F.S中，而细胞周期蛋白E.细胞周期蛋白A.cdk2仅存在于E2F.S复合物中。这些数据表明，在G1晚期从MT2位点去除含p107的E2F.G0/G1复合物（一种候选阻遏物）可能对小鼠TK基因的S期依赖性转录至关重要。
