细胞动粒蛋白Hec1启动子的结构及功能分析  

Structure and Function Analyses of Kinetochoric Protein Hec1 Promoter

作　　者：李良维[1] 刘兢[1] 姚雪彪[1] 程联胜[1] 

机构地区：[1]中国科学技术大学生命科学学院细胞免疫学实验室,合肥230027

出　　处：《中国生物化学与分子生物学报》2006年第11期869-875,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.30400078);国家重点基础研究发展规划(973计划;No.2002CB713700);安徽省自然科学基金资助课题(No.050430201)~~

摘　　要：细胞的分裂是一个严格调控,高度有序的过程.为了将复制后的染色体均匀、准确地传递给两个子细胞,细胞在分裂中后期受到纺锤体检验点的严格监控.Hec1定位于动粒,是纺锤体检验点调控的关键蛋白之一,它通过螺旋-螺旋结构域与其他动粒蛋白相互作用调节姐妹染色体的精确分离.为研究Hec1转录水平的调控机理,采用BLAST工具,从GenBank中搜索到了人Hec1基因上游的序列,并利用在线工具http://mbs.cbrc.jp/research/db/TFSEARCH.html提供的转录因子结合位点搜索引擎,对其5′启动子调节区段进行了分析.分析结果表明:在Hec1基因上游-200～-1序列内,存在E2F、ATF4和cAMP应答元件结合蛋白(CREB)等转录因子调控元件.在结构分析的基础上,提取HeLa细胞基因组DNA,用PCR方法克隆了Hec1基因启动子,并构建了多个含启动子不同区段的pGL3荧光素酶报告基因表达质粒.瞬时转染HeLa细胞后的结果表明,-70～-63以及-155～-144之间的启动子区对维持荧光素酶活性最为关键.凝胶迁移实验证明,这两个区段分别能够和转录因子CREB以及ATF4结合.随后,采用野生型的以及含有133位磷酸化位点突变的CREB转染HeLa细胞,通过荧光定量PCR实验发现,Hec1的表达水平分别出现明显上升和下降.该结果表明,Hec1表达的调控是通过CREB的活化来完成的.Cell mitosis is a highly ordered and precisely regulated process. To distribute the two replicated sister chromatids into two daughter cells equally, cell cycle progression is supervised strictly by spindle checkpoint during metaphase to anaphase. Hecl localizes to kinetochore in cell mitosis, and it is the critical protein for M phase progression. Hecl interacts with other kinetochorlc proteins through its coiled-coil domain to regulate the accurate sister chromatid separation. To clarify the mechanism of transcriptional regulation of Hecl gene, we searched out the human Hecl gene sequence from GenBank using BLAST tool and its 5＇ flank region was analyzed using promoter analysis software online. The result showed that Hec 1 5＇ upstream - 200 ～ - 1 bp region contained the putative transcription factor recognition sites including E2F, activating transcription factor 4 （ATF4）, and cAMP response element binding protein （CREB）. Subsequently, we extracted the genomic DNA from HeLa cells, amplified and cloned the promoter region by PCR, and constructed a series of pGL3 luciferase reporter vectors which contained different sections of Hec 1 5＇-upstream region. The results from HeLa cell transient transfection demonstrated that the seauences between - 70 ～ - 63 bp and - 155 ～ - 144 bp upstream of translational start site is critical for transcriptional activity. Gel shift and supershift assays also demonstrated specific binding of AFT4 and CREB proteins to their putative boxes. The expression of Hecl mRNA was up- or down-regulated by overexpression of CREB or its Ser-133 to Ala dominant negative mutant, respectively. These results suggested that the Hecl expression could be regulated through activation of transcriptional factor CREB.

关 键 词：Hecl 动粒 启动子 活化转录因子4 cAMP应答元件结合蛋白 

分 类 号：Q78[生物学—分子生物学] R394[医药卫生—医学遗传学]