Sp1 and Sp3 are involved in the full transcriptional activity of centromere protein H in human nasopharyngeal carcinoma cells
Sp1 and Sp3 are involved in the full transcriptional activity of centromere protein H in human nasopharyngeal carcinoma cells
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DOI：
10.1111/j.1742-4658.2012.08654.x
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发表时间：
2012-08
期刊：
影响因子：
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通讯作者：
Wei-feng Zhao;Hong-bo Wang;B. Xie;Li‐juan Hu;Li-Hua Xu;B. Kuang;Man‐zhi Li;Xing Zhang
中科院分区：
文献类型：
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作者：
Wei-feng Zhao;Hong-bo Wang;B. Xie;Li‐juan Hu;Li-Hua Xu;B. Kuang;Man‐zhi Li;Xing Zhang
着丝粒蛋白H（CENPH）是人类活动着丝粒的基本组成部分之一，其过表达已被证明与人类癌症密切相关。但其转录调控机制尚未见报道。本研究的目的是研究鼻咽癌细胞中CENPH的转录调控元件。为了表征CENPH启动子并鉴定调控元件，我们从永生化正常鼻咽上皮细胞（Bmi-1/NPEC）中克隆了CENPH基因5′侧翼区的1015 bp（−975/+40 bp）。功能分析确定了参与调节人CENPH启动子活性的最小区域（−140/−87 bp）。通过定点突变、反式激活试验、染色质免疫沉淀和电泳迁移率变动试验，我们发现Sp1/Sp3转录因子在体外和体内都可以与CENPH启动子结合，并且它们调节人鼻咽癌细胞中CENPH启动子的激活。Sp1和Sp3在鼻咽癌细胞中高表达。小干扰RNA敲低Sp1和Sp3或光神霉素A抑制Sp1和Sp3活性可降低CENPH mRNA表达，而外源性Sp1和Sp3表达可上调CENPH mRNA表达。两者合计，我们的研究结果表明，Sp1和Sp3结合到CENPH最小启动子和功能作为一个调节器的转录CENPH在人鼻咽癌。
The overexpression of centromere protein H (CENPH), one of the fundamental components of the human active kinetochore, has been shown to be closely associated with human cancers. However, the mechanism of its transcriptional regulation has not been reported. The aim of the present study was to investigate the regulatory elements for the transcriptional regulation of CENPH in nasopharyngeal carcinoma cells. To characterize the CENPH promoter and identify regulatory elements, we cloned 1015 bp (−975/+40 bp) of the 5′‐flanking region of the CENPH gene from immortalized normal nasopharyngeal epithelial cells (Bmi‐1/NPEC). Functional analysis established a minimal region (−140/−87 bp) involved in the regulation of human CENPH promoter activity. Through site‐directed mutagenesis, a transactivation assay, chromatin immunoprecipitation, and electrophoretic mobility shift assay, we found that the Sp1/Sp3 transcription factors could bind to the CENPH promoter in vitro and in vivo, and that they regulated CENPH promoter activation in human nasopharyngeal carcinoma cells. Furthermore, Sp1 and Sp3 were highly expressed in nasopharyngeal carcinoma cells. Knockdown of Sp1 and Sp3 by small interfering RNA or inhibition of Sp1 and Sp3 activity by mithramycin A decreased CENPH mRNA expression, whereas the exogenous expression of Sp1 and Sp3 upregulated CENPH mRNA expression. Taken together, our results indicate that Sp1 and Sp3 bind to the CENPH minimal promoter and function as a regulator of the transcription of CENPH in human nasopharyngeal carcinomas.