Functional characterization of the promoter of human kinetochore protein HEC1: Novel link between regulation of the cell cycle protein and CREB family transcription factors

Liansheng Cheng, Liangwei Li, Xinxian Qiao, Jing Liu, Xuebiao Yao
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引用次数: 11

Abstract

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.

人着丝点蛋白HEC1启动子的功能表征:细胞周期蛋白调控与CREB家族转录因子之间的新联系
HEC1(在癌症中高表达)在细胞有丝分裂中定位于着丝点,在染色体分离中起着至关重要的作用。为了阐明其转录调控机制,我们对其5 '侧区进行了筛选和分离。该区域的定位鉴定出它是一个TATA-less启动子,并包含几个不同转录因子的假定结合位点。用含有HEC1 5 '侧区渐进缺失的pGL3荧光素酶报告载体转染HeLa细胞的结果表明,含有cAMP响应元件结合蛋白(CREB)和激活转录因子4 (ATF4或CREB2)结合位点的两个元件对转录活性至关重要。这两个元件的突变,而不是下游的E2F盒子,导致启动子活性显著降低。凝胶移位和超移位实验也证明了转录因子对其推测的结合位点的特异性结合。此外,CREB或ATF4的过表达增强了HEC1启动子的激活,并且两者的过表达对HEC1转录的激活具有叠加效应。相反,CREB或ATF4显性阴性突变体的过表达会导致HEC1 mRNA的显著下调。我们的研究为CREB家族转录因子参与癌相关细胞中着丝点蛋白HEC1调控的潜在机制提供了新的见解。
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