Liu Huaying, Zhou Ming, Luo Xiaomin, Zhang Liming, Niu Zhaoxia, Peng Cong, Ma Jian, Peng Shuping, Zhou Houde, Xiang Bo, Li Xiayu, Li Shufang, He Jiajin, Li Xiaoling, Li Guiyuan
Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha, Hunan 410078, People's Republic of China.
BMC Mol Biol. 2008 Dec 27;9:111. doi: 10.1186/1471-2199-9-111.
Bromodomain is an evolutionally conserved domain that is found in proteins strongly implicated in signal-dependent transcriptional regulation. Genetic alterations of bromodomain genes contributed to the development of many human cancers and other disorders. BRD7 is a recently identified bromodomain gene. It plays a critical role in cellular growth, cell cycle progression, and signal-dependent gene expression. Previous studies showed that BRD7 gene exhibited much higher-level of mRNA expression in normal nasopharyngeal epithelia than in nasopharyngeal carcinoma (NPC) biopsies and cell lines. However, little is known about its transcriptional regulation. In this study, we explored the transcriptional regulation of BRD7 gene.
Potential binding sites of transcription factors within the promoter region of BRD7 gene were predicted with MatInspector Professional http://genomatix.de/cgi-bin/matinspector_prof/mat_fam.pl. Mutation construct methods and luciferase assays were performed to define the minimal promoter of BRD7 gene. RT-PCR and western blot assays were used to detect the endogenous expression of transcription factor Sp1, c-Myc and E2F6 in all cell lines used in this study. Electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) were used to detect the direct transcription factors that are responsible for the promoter activity of BRD7 gene. DNA vector-based siRNA technology and cell transfection methods were employed to establish clone pools that stably expresses SiRNA against c-Myc expression in nasopharyngeal carcinoma 5-8F cells. Real-time PCR was used to detect mRNA expression of BRD7 gene in 5-8F/Si-c-Myc cells.
We defined the minimal promoter of BRD7 gene in a 55-bp region (from -266 to -212bp), and identified that its promoter activity is inversely related to c-Myc expression. Sp1 binds to the Sp1/Myc-Max overlapping site of BRD7 minimal promoter, and slightly positively regulate its promoter activity. c-Myc binds to this Sp1/Myc-Max overlapping site as well, and negatively regulates the promoter activity and endogenous mRNA expression of BRD7 gene. Knock-down of c-Myc increases the promoter activity and mRNA level of BRD7 gene. The luciferase activity of the mutated promoter constructs showed that Sp1/Myc-Max overlapping site is a positive regulation element of BRD7 promoter.
These studies provide for the first time the evidence that c-Myc is indeed a negative regulator of BRD7 gene. These findings will help to further understand and uncover the bio-functions of BRD7 gene involved in the pathogenesis of NPC.
溴结构域是一个进化上保守的结构域，存在于与信号依赖转录调控密切相关的蛋白质中。溴结构域基因的遗传改变与许多人类癌症及其他疾病的发生发展有关。BRD7是最近鉴定出的一个溴结构域基因。它在细胞生长、细胞周期进程及信号依赖基因表达中起关键作用。先前研究表明，BRD7基因在正常鼻咽上皮中的mRNA表达水平远高于鼻咽癌活检组织和细胞系。然而，其转录调控机制鲜为人知。在本研究中，我们对BRD7基因的转录调控进行了探索。
我们确定了BRD7基因的最小启动子位于一个55bp区域（从-266至-212bp），并发现其启动子活性与c-Myc的表达呈负相关。Sp1结合到BRD7最小启动子的Sp1/Myc-Max重叠位点，并对其启动子活性有轻微的正向调节作用。c-Myc也结合到该Sp1/Myc-Max重叠位点，并对BRD7基因启动子活性和内源性mRNA表达起负向调节作用。敲低c-Myc可增加BRD7基因的启动子活性和mRNA水平。突变启动子构建体的荧光素酶活性表明，Sp1/Myc-Max重叠位点是BRD7启动子的一个正向调控元件。
这些研究首次提供证据表明c-Myc确实是BRD7基因的负调控因子。这些发现将有助于进一步理解和揭示BRD7基因在鼻咽癌发病机制中的生物学功能。
