Hayashi Y, Hirose F, Nishimoto Y, Shiraki M, Yamagishi M, Matsukage A, Yamaguchi M
Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan.
J Biol Chem. 1997 Sep 5;272(36):22848-58. doi: 10.1074/jbc.272.36.22848.
The Drosophila proliferating cell nuclear antigen (PCNA) gene promoter contains at least three transcriptional regulatory elements, the URE (upstream regulatory element), DRE (DNA replication-related element), and E2F recognition sites. In nuclear extracts of Drosophila Kc cells, we detected a novel protein factor(s), CFDD (common regulatory factor for DNA replication and DREF genes) that appeared to recognize two unique nucleotide sequences (5'-CGATA and 5'-CAATCA) and bind to three sites in the PCNA gene promoter. These sites were located at positions -84 to -77 (site 1), -100 to -93 (site 2) and -134 to -127 (site 3) with respect to the transcription initiation sites. Sites 2 and 3 overlapped with DRE and URE, respectively, and the 5'-CGATA matched with the reported recognition sequence of BEAF-32 (boundary element-associated factor of 32 kDa). Detailed analyses of CFDD recognition sequences and experiments with specific antibodies to DREF (DRE-binding factor) and BEAF-32 suggest that CFDD is different from DREF, UREF (URE-binding factor) and BEAF-32. A UV cross-linking experiment revealed that polypeptides of approximately 76 kDa in the nuclear extract interact directly with the CFDD site 1 sequence. Transient expression assays of chloramphenicol acetyltransferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying mutations in CFDD site 1 and examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying mutations in site 1, introduced into flies by germ line transformation, revealed that CFDD site 1 plays an important role for the promoter activity both in cultured cells and in living flies. In addition to the PCNA gene, multiple CFDD sites were found in promoters of the DNA polymerase alpha and DREF genes, and CFDD binding to the DREF promoter was confirmed. Therefore, CFDD may play important roles in regulation of Drosophila DNA replication-related genes.
果蝇增殖细胞核抗原（PCNA）基因启动子包含至少三个转录调控元件，即上游调控元件（URE）、DNA复制相关元件（DRE）和E2F识别位点。在果蝇Kc细胞的核提取物中，我们检测到一种新的蛋白质因子，即DNA复制和DREF基因的共同调控因子（CFDD），它似乎能识别两个独特的核苷酸序列（5'-CGATA和5'-CAATCA），并与PCNA基因启动子中的三个位点结合。相对于转录起始位点，这些位点分别位于-84至-77位（位点1）、-100至-93位（位点2）和-134至-127位（位点3）。位点2和位点3分别与DRE和URE重叠，且5'-CGATA与已报道的32 kDa边界元件相关因子（BEAF-32）的识别序列匹配。对CFDD识别序列的详细分析以及使用针对DREF（DRE结合因子）和BEAF-32的特异性抗体进行的实验表明，CFDD与DREF、URE结合因子（UREF）和BEAF-32不同。紫外线交联实验表明，核提取物中约76 kDa的多肽直接与CFDD位点1序列相互作用。用携带CFDD位点1突变的PCNA启动子-CAT融合基因转染Kc细胞后进行氯霉素乙酰转移酶（CAT）的瞬时表达分析，并检测通过种系转化导入果蝇的携带位点l突变的PCNA启动子-lacZ融合基因的lacZ表达，结果表明CFDD位点1对培养细胞和活体果蝇中的启动子活性均起着重要作用。除了PCNA基因外，在DNA聚合酶α和DREF基因的启动子中还发现了多个CFDD位点，并且证实了CFDD与DREF启动子的结合。因此，CFDD可能在果蝇DNA复制相关基因的调控中发挥重要作用。
