Functional characterization of the promoter of human kinetochore protein HEC1: Novel link between regulation of the cell cycle protein and CREB family transcription factors
Functional characterization of the promoter of human kinetochore protein HEC1: Novel link between regulation of the cell cycle protein and CREB family transcription factors
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DOI：
10.1016/j.bbaexp.2007.07.005
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发表时间：
2007-09-01
期刊：
影响因子：
--
通讯作者：
Yao, Xuebiao
中科院分区：
文献类型：
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作者：
Cheng, Liansheng;Li, Liangwei;Yao, Xuebiao
HEC1(在肿瘤中高表达)在细胞有丝分裂中定位于着丝粒，在M期进展的染色体分离中起着重要作用。为了阐明其转录调控机制，我们搜索并分离了其5‘侧翼区。对该区域的定位表明，它是一个不含TATA的启动子，并且含有几个可能与不同转录因子结合的位点。结果表明，含有cAMP反应元件结合蛋白(CREB)结合位点和激活转录因子4(ATF4或CREB2)的两个元件对转录活性起关键作用。这两个元件的突变，而不是下游的E2F盒，导致启动子活性显著降低。凝胶移位和超移位实验也证明转录因子与它们可能的结合部位有特异性结合。此外，CREB或ATF4的过表达增强了HEC1启动子的激活，两者的过表达对HEC1转录的激活具有相加作用。相反，CREB或ATF4的显性负性突变体的过表达导致HEC1基因的表达显著下调。我们的研究为CREB家族转录因子如何参与动粒蛋白HEC1在肿瘤相关细胞中的调控提供了新的机制。(C)2007爱思唯尔B.V保留所有权利。
HEC1 (highly expressed in cancer), which localizes to kinetochore in cell mitosis, plays an essential role in chromosome segregation for M phase progression. To clarify the mechanism of its transcriptional regulation, we searched out and isolated its 5 '-flanking region. Mapping of this region identified that it is a TATA-less promoter and contains several putative binding sites for different transcription factors. The results from HeLa cells transfected with pGL3 luciferase reporter vectors containing progressive deletion of the HEC1 5 '-flanking region demonstrated that two elements containing binding sites for cAMP responsive element binding (CREB) protein and activating transcription factor 4 (ATF4 or CREB2) are critical for transcriptional activity. Mutation of the two elements, not downstream E2F box, resulted in a significant reduction of the promoter activity. Gel shift and supershift assays also demonstrated specific binding of transcription factors to their putative binding sites. Furthermore, overexpression of either CREB or ATF4 enhanced the activation of the HEC1 promoter and overexpression of both of them had an additive effect on the activation of the HEC1 transcription. Conversely, overexpression of dominant negative mutants of either CREB or ATF4 resulted in downregulation of HEC1 mRNA significantly. Our study provided a new insight into a potential mechanism of how transcription factors of CREB family are involved in the regulation of kinetochore protein HEC1 in cancer-related cells. (c) 2007 Elsevier B.V All rights reserved.