HeLa细胞ezrin基因基本启动子区转录调控元件的鉴定

 目的 鉴定HeLa细胞中调控ezrin基因基本启动子活性的顺式作用元件,探讨ezrin基因在HeLa细胞的表达调控机制。 方法 采用碱基定点突变实验和双荧光素酶报告基因分析系统,检测在HeLa细胞中Sp1结合位点(-75/-69, 相对于转录起始位点)和AP 1结合位点(-64/-58)对人ezrin基因基本启动子活性的影响;采用电泳迁移率变动分析法(EMSA)实验,检测ezrin基因基本启动子区序列与HeLa细胞核蛋白提取物的特异性结合活性。 结果 在HeLa细胞中,单独删除和置换Sp1结合位点或AP 1结合位点,ezrin基因基本启动子活性降低50％左右;同时置换Sp1结合位点和AP 1结合位点,启动子活性几乎完全丧失。ezrin基因基本启动子序列能够与HeLa细胞核蛋白提取物相结合。 结论 HeLa细胞中,Sp1结合位点和AP 1结合位点为ezrin基因基本启动子区的重要转录调控元件,有可能存在某种转录因子与之结合,激活ezrin基因转录。     

食管癌细胞Ezrin基因转录调控区克隆及其启动子活性的鉴定

目的:克隆食管癌侵袭转移相关基因Ezrin的转录调控区序列,进行启动子活性鉴定及初步的生物信息学分析。方法:利用在线程序对Ezrin基因可能的转录调控区进行GC含量和CpG岛分析以及转录因子结合位点预测;采用PCR法从食管癌细胞基因组DNA中克隆Ezrin基因-1759/ 134和-726/ 134区段,构建萤火虫荧光素酶报告基因表达质粒pGLB-hE(-1759/ 134)和pGLB-hE(-726/ 134),检测所克隆片段的启动子活性。结果:Ezrin基因5′侧翼区为高GC含量区,存在CpG岛,无典型的TATA盒,然而转录因子Sp1结合位点却无处不在。与对照质粒pGLB相比,重组质粒pGLB-hE(-726/ 134)具有较强的荧光素酶活性,pGLB-hE(-1759/ 134)的荧光素酶活性约是pGLB-hE(-726/ 134)的2倍。结论:Ezrin基因的-726/ 134区段具有启动子活性,含有Ezrin基因的核心启动子区和调节性启动子区;-1759/-726区段具有增强启动子活性的作用,含有Ezrin基因增强子元件区。     

人卵巢癌相关候选基因HE4(WFDC2)的转录调控主要由Sp1与位于-71和-48的Egr-1位点结合所介导

目的阐明HE4基因转录调控机制的细节.方法1.用半定量RT-PCR的方法评估HE4基因在肿瘤细胞株中的表达情况;2.以荧光素酶报告基因载体为基础,对HE4基因的上游顺序构建了3'、5'缺失突变和一系列连接体扫描突变;3.通过瞬时转染/体外双荧光素酶报告系统对这些重组质粒中插入片段进行启动子活性的分析;4.针对蛋白和关键顺式区域的结合的特异性和能力,开展泳动滞后和抗体介导的超迁移分析.结果通过对(-1860/ 29)的HE4基因上游片段及其剪切体的分析,将HE4基因最小启动子确定为-107/ 15的DNA片段.通过对连接体扫描突变体的分析,将关键的顺式作用元件确定在W45(-71/-48)片段,生物信息学提示该区存在两个Egr-1位点.通过用已知的反式作用因子与报告基因质粒分别进行共转染,提示Sp1是最为有效的反式作用因子,而不是Egr-1.通过泳动滞后和抗体介导的超迁移实验,证实Sp1确实是参与作用的转录因子.结论HE4基因的转录活性主要是由转录因子Sp1与位于(-71/-48)区域的两个Egr-1位点结合所介导的.     

Genomic structure, chromosomal mapping, and promoter region analysis of murine uridine phosphorylase gene.

Uridine phosphorylase (UPase) plays an important role in the activation of 5-fluorouracil and in the regulation of tissue and plasma concentration of uridine, a potential biochemical modulator of 5-fluorouracil therapy. UPase expression is affected by the c-H-ras oncogene and various cytokines through unknown mechanisms. To understand its expression and regulation, we cloned the murine UPase gene, defined its genomic organization, determined its 5'- and 3'-end flanking sequences, and evaluated the promoter activity. The UPase gene contains nine exons and eight introns, spanning a total of approximately 18.0 kb. Its promoter lacks canonical TATA and CCAAT boxes, although a CAATAAAAA TATA-like box is seen from -41 to -49. Furthermore, IFN regulatory factor 1, c/v-Myb, and p53 binding sites are present in the promoter region, indicating that UPase expression may be directly regulated by cytokines and oncogene products. The 1.2-kb flanking fragment showed promoter activity driving the expression of the luciferase gene in various mammalian cells. A TGGGG repeat sequence is seen in the 3'-end flanking region. This element is considered to be a potential recombination consensus hot spot that may contribute to the encoding of different UPase isoforms present in different tissues, both normal and neoplastic.     

Ets transcription factors cooperate with Sp1 to activate the human tenascin-C promoter

Tenascin-C (TN-C), an extracellular matrix glycoprotein is expressed during embryonic development, but is present only at low levels in normal adult tissues. TN-C is re-expressed during wound healing, fibrotic diseases and in cancer. To better understand the mechanisms that control TN-C gene expression, we examined the regulation of the human TN-C promoter in human fibroblasts. We demonstrate that a short segment of the TN-C promoter between bp -133 and -27 contains three evolutionarily conserved Ets binding sites (EBS). These three EBSs bind in vitro expressed Fli1 protein and mediate transactivation of the TN-C gene by Fli1. Furthermore, two proximal EBSs contribute significantly to basal activity of the TN-C promoter. GABP, which is present in human fibroblast nuclear extracts, interacts with the two proximal EBSs. In addition, several Sp1 and Sp3 binding sites have been located in close proximity to the EBSs within this promoter region. The studies performed in Drosophila cells demonstrate that either Fli1 or GABPalpha+beta1 functionally interact with Sp1 resulting in a synergistic stimulation of the TN-C promoter activity. In conclusion, this study shows for the first time that the TN-C gene is regulated by Ets proteins, which together with Sp1 act as potent activators of TN-C expression.