Yamaguchi M, Hayashi Y, Matsukage A
Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Nagoya, Japan.
J Biol Chem. 1995 Oct 20;270(42):25159-65. doi: 10.1074/jbc.270.42.25159.
We have found sequences similar to the transcription factor E2F recognition site within the Drosophila proliferating cell nuclear antigen (PCNA) gene promoter. These sequences are located at positions -43 to -36 (site I). and -56 to -49 (site II) with respect to the cap site Glutathione S-transferase (GST)-E2F and GST-DP fusion proteins cooperate and bind to the potential E2F sites in the PCNA promoter in vitro. A binding factor(s) to these sequences that has similar binding specificity to that of E2F was detected in nuclear extracts of Drosophila Kc cells. Furthermore, transient expression of target site for the activation coincided with the E2F sites. These results indicate that the PCNA gene is a likely target gene of E2F. Examination of lacZ expression from PCNA-lacZ fusion genes carrying mutations in either or both of two E2F sites introduced into flies by germ line transformation revealed that site II plays a major role in the PCNA promoter activity during embryogenesis and larval development, although both sites are required for optimal promoter activity. However, for maternal expression in ovaries, either one of the two sites is essentially sufficient to direct optimal promoter activity. These results demonstrate, for the first time, an essential role for E2F sites in regulation of PCNA promoter activity during development of a multicellular organism.
我们在果蝇增殖细胞核抗原（PCNA）基因启动子内发现了与转录因子E2F识别位点相似的序列。这些序列相对于帽位点位于-43至-36位（位点I）和-56至-49位（位点II）。谷胱甘肽S-转移酶（GST）-E2F和GST-DP融合蛋白在体外协同作用并结合PCNA启动子中的潜在E2F位点。在果蝇Kc细胞核提取物中检测到一种与这些序列结合的因子，其结合特异性与E2F相似。此外，激活靶位点的瞬时表达与E2F位点一致。这些结果表明PCNA基因可能是E2F的靶基因。通过种系转化将携带两个E2F位点中一个或两个发生突变的PCNA-lacZ融合基因导入果蝇，对lacZ表达的检测表明，尽管两个位点对于最佳启动子活性都是必需的，但位点II在胚胎发生和幼虫发育过程中对PCNA启动子活性起主要作用。然而，对于卵巢中的母体表达，两个位点中的任何一个基本上足以指导最佳启动子活性。这些结果首次证明了E2F位点在多细胞生物体发育过程中对PCNA启动子活性调节的重要作用。
