Transcriptional regulation of the Drosophila rfc1 gene by the DRE-DREF pathway
Transcriptional regulation of the Drosophila rfc1 gene by the DRE-DREF pathway
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DOI：
10.1111/j.1742-4658.2007.05730.x
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发表时间：
2007-04-01
期刊：
影响因子：
5.4
通讯作者：
Yamaguchi, Masamitsu
中科院分区：
文献类型：
--
作者：
Tsuchiya, Akihiro;Inoue, Yoshihiro H.;Yamaguchi, Masamitsu
DNA复制相关元件(DRE)是在许多DNA复制相关基因的启动子中发现的一个常见的8个碱基的序列(5‘-TATCGATA)，DRE结合因子(DREF)特异性地与其结合以激活转录。复制因子C(RFC)是DNA复制过程中必不可少的5个亚基复合体，其中最大的亚基是RFC140。我们首先鉴定了编码果蝇RFC140(DRFC140)蛋白的基因(Rfc1)，然后分离到一个突变体。这些表型表明，该基因对细胞周期进程是必不可少的，免疫细胞化学研究也表明其表达与细胞周期之间存在关系。Rfc1基因在其5‘侧翼区包含三个类似Dre的序列，其中一个与Dre完全匹配，另外两个在八个核苷酸中的七个核苷酸上完全匹配。这些序列分别命名为DRE1(-63至-69)、DRE2(-378至-385)和DRE3(-1127至-1134)。用抗DREF血清对三龄幼虫多线染色体进行免疫染色，在3R染色体的82e2处检测到一条含有Rfc1基因区的特异性条带。用果蝇KC细胞核提取物进行的条带迁移率分析表明，DREF在体外与DRE1、-2和-3结合，用抗DREF抗体进行染色质免疫沉淀证实这在体内发生了。S2细胞中荧光素酶的瞬时表达分析进一步表明，Rfc1启动子中的DRE参与了该基因的转录调控。此外，在DREF双链RNA处理的S2细胞中，Rfc1启动子的活性降低了38%。这些结果表明，DREF对Rfc1启动子有正向调节作用。
The DNA replication-related element (DRE) is a common 8-bp sequence (5'-TATCGATA) found in the promoters of many DNA replication-related genes, to which DRE-binding factor (DREF) specifically binds to activate transcription. Replication factor C (RFC) is an essential five-subunit complex in DNA replication, the largest subunit being RFC140. We first identified the gene (rfc1) encoding the Drosophila RFC140 (dRFC140) protein and then isolated a mutant. The phenotypes suggested that the gene is essential for cell-cycle progression, and immunocytochemical studies also indicated a relation between its expression and the cell cycle. The rfc1 gene contains three DRE-like sequences in its 5'-flanking region, one of them perfectly matching DRE and the other two demonstrating a match in seven of eight nucleotides. These sequences were named DRE1 (-63 to -69), DRE2 (-378 to -385), and DRE3 (-1127 to -1134), respectively. Immunostaining of polytene chromosomes in third-instar larvae using anti-DREF sera detected a specific band in 82E2 of 3R chromosome, containing the rfc1 gene region. Band-mobility shift assays using Drosophila Kc cell nuclear extracts revealed that DREF binds to DRE1, -2, and -3 in vitro, and chromatin immunoprecipitation using anti-DREF IgG confirmed that this occurs in vivo. Luciferase transient expression assays in S2 cells further suggested that DREs in the rfc1 promoter are involved in transcriptional regulation of the gene. Moreover, rfc1 promoter activity was reduced by 38% in DREF double-stranded RNA-treated S2 cells. These results indicate that DREF positively regulates the rfc1 promoter.