Antagonistic regulation of the Drosophila PCNA gene promoter by DREF and Cut
Antagonistic regulation of the Drosophila PCNA gene promoter by DREF and Cut
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DOI：
10.1111/j.1365-2443.2006.00956.x
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发表时间：
2006-05-01
期刊：
影响因子：
2.1
通讯作者：
Yamaguchi, M
中科院分区：
文献类型：
--
作者：
Seto, H;Hayashi, Y;Yamaguchi, M
果蝇增殖细胞核抗原（dPCNA）基因启动子含有多个转录调控元件，如上游调控元件（URE）、DNA复制相关元件（DRE，5 '-TATCGATA）和E2 F识别位点。在本研究中，酵母单杂交筛选使用dPCNA启动子中的DRE的三个串联重复序列作为诱饵，允许分离编码Cut的cDNA，Cut是哺乳动物CCAAT置换蛋白（CDP）/Cux的果蝇同源物。电泳迁移率变动分析表明，Cut与DRE和URE中的5 '-AATCAAAC序列都结合，对前者的亲和力要高得多。通过瞬时荧光素酶表达测定法在果蝇S2细胞中对切割或DREF进行RNA干扰后测量dPCNA启动子活性，显示DREF激活dPCNA启动子，而切割作为阻遏物发挥功能。染色质免疫沉淀分析在20-羟基蜕皮激素的存在或不存在下进一步显示DREF和切蛋白定位在基因组区域中含有的dPCNA启动子在S2细胞中，特别是在切的情况下诱导分化。这些结果表明，在分化状态下，Cut通过与启动子区结合而作为dPCNA基因的转录抑制因子发挥作用，而DREF在细胞增殖期间与DRE结合以促进dPCNA的表达。
The gene promoter of Drosophila proliferating cell nuclear antigen (dPCNA) contains several transcriptional regulatory elements, such as upstream regulatory element (URE), DNA replication-related element (DRE, 5'-TATCGATA), and E2F recognition sites. In the present study, a yeast one-hybrid screen using three tandem repeats of DRE in dPCNA promoter as the bait allowed isolation of a cDNA encoding Cut, a Drosophila homolog of mammalian CCAAT-displacement protein (CDP)/Cux. Electrophoretic mobility shift assays showed that Cut bound to both DRE and the sequence 5'-AATCAAAC in URE, with much higher affinity to the former. Measurement of dPCNA promoter activity by transient luciferase expression assays in Drosophila S2 cells after an RNA interference for Cut or DREF showed DREF activates the dPCNA promoter while Cut functions as a repressor. Chromatin immunoprecipitation assays in the presence or absence of 20-hydroxyecdysone further showed both DREF and Cut proteins to be localized in the genomic region containing the dPCNA promoter in S2 cells, especially in the Cut case upon induction of differentiation. These results indicate that Cut functions as a transcriptional repressor of dPCNA gene by binding to the promoter region in the differentiated state, while DREF binds to DRE to promote expression of dPCNA during cell proliferation.