Multiple NF-Y-binding CCAAT boxes are essential for transcriptional regulation of the human C7orf24 gene, a novel tumor-associated gene
Multiple NF-Y-binding CCAAT boxes are essential for transcriptional regulation of the human C7orf24 gene, a novel tumor-associated gene
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DOI：
10.1111/j.1742-4658.2011.08314.x
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发表时间：
2011-11-01
期刊：
影响因子：
5.4
通讯作者：
Kakeya, Hideaki
中科院分区：
文献类型：
--
作者：
Ohno, Yuji;Hattori, Akira;Kakeya, Hideaki
人7号染色体ORF 24（C7 orf 24）已被鉴定为肿瘤相关蛋白，并且显示为c-谷氨酰环转移酶。本研究对人C7 orf 24基因的启动子区进行了分析，以探讨该基因的转录调控机制。我们发现，人类C7 orf 24启动子是一个TATA-少启动子，含有5个CCAAT盒在一个正向排列。通过在HeLa、MCF-7和IMR-90细胞中用5 '缺失和定点突变的构建体进行荧光素酶报告基因测定，我们发现三个近端CCAAT盒对于基础转录是重要的。电泳迁移率凝胶迁移试验和染色质免疫沉淀试验表明，NF-Y特异性结合所有三个CCAAT盒。此外，C7 orf 24的mRNA和蛋白质表达水平显着降低，在HeLa细胞的NF-YB，NF-Y的亚基耗尽。这些结果表明，结合到三个近端CCAAT盒的NF-γ在该基因的转录中起核心作用。此外，在多个NF-Y的控制下转录的其他基因的情况下，如人E2 f1和细胞周期蛋白b1，C7 orf 24基因的表达谱在细胞周期中振荡，这意味着C7 orf 24是一个新的细胞周期相关基因。
Human chromosome 7 ORF 24 (C7orf24) has been identified as a tumor-related protein, and shown to be a c-glutamyl cyclotransferase. In the current study, we characterized the promoter region of the human C7orf24 gene to explore the transcriptional regulation of the gene. We revealed that the human C7orf24 promoter is a TATA-less promoter, containing five CCAAT boxes aligned in a forward orientation. By performing a luciferase reporter assay with 5'-deleted and site-directed mutated constructs in HeLa, MCF-7 and IMR-90 cells, we found that three proximal CCAAT boxes are important for basal transcription. Electrophoretic mobility gel shift assay and chromatin immunoprecipitation assay demonstrated that NF-Y specifically bound to all three CCAAT boxes. In addition, the mRNA and protein expression levels of C7orf24 were significantly reduced in HeLa cells depleted of NF-YB, a subunit of NF-Y. These results suggested that NF-Ys bound to the three proximal CCAAT boxes play a central role in the transcription of the gene. Furthermore, as in the case of other genes transcribed under the control of multiple NF-Ys, such as human E2f1 and cyclin b1, the C7orf24 gene expression profile oscillated during the cell cycle, implying that C7orf24 is a novel cell cycle-associated gene.