人成纤维细胞转录因子Sp1Sp3对p16^(INK4a)基因的调控  

The Role of Transcription Factor Sp1/Sp3 in the Regulation of p16^(INK4a) Gene in Human Diploid Fibroblasts

作　　者：吴军峰[1] 童坦君[1] 张宗玉[1] 

机构地区：[1]北京大学医学部生物化学与分子生物学系,北京100083

出　　处：《中国生物化学与分子生物学报》2005年第1期88-93,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重点基础研究发展规划 (No .G2 0 0 0 0 5 70 0 1);国家自然科学基金项目 (No .3 0 2 714 3 2;3 0 3 715 61)资助课题~~

摘　　要：p16 INK4a是一种细胞周期蛋白依赖激酶 (cdk)的抑制因子 ,它通过抑制cdk4与cdk6的活性 ,使视网膜母细胞瘤抑制蛋白Rb处于低磷酸化状态 ,从而使细胞阻滞于G1期 .对p16 INK4aATG上游 6 2 2bp片段进行序列分析发现 ,该区域富含GC ,其中有 5个GC盒 (分别命名为GC Ⅰ～GC Ⅴ ) .将上述片段插入到荧光素酶报告载体pGL3 Basic ,分别对 5个GC盒进行点突变后转染人胚肺二倍体成纤维细胞 (2BS)发现 ,Ⅰ、Ⅱ、Ⅳ位点的突变体显著下调p16 INK4a启动子的活性 ,而Ⅲ、Ⅴ位点突变体无明显作用 .电泳迁移率变动分析 (EMSA)证实 ,GC Ⅰ ,Ⅱ ,Ⅳ能与转录因子Sp1和Sp3结合 ,而且结合条带可被转录因子Sp1和Sp3的抗体所拮抗 .共转染Sp1有助于增加启动子的活性 ,而共转染Sp3则有较弱的抑制作用 ,证明p16 INK4a的转录受到Sp1与Sp3的调控 .p16 INK4a is one of the inhibitors of cyclin dependent kinase(cdk),which inhibits cdk4/cdk6 mediated phosphorylation of retinoblastoma gene product (Rb) that leads to cell cycle arrest in G 1 phase. The 622 bp fragment in upstream transcription start site of p 16 INK4a was analyzed and it was found that the fragment was rich in GC and contained five GC boxes(named GC I～GC V). The fragment was inserted into the reporter vector pGL3 Basic, and the each of five GC boxes were mutated respectively. These constructs were transiently transfected into the human embryonic lung diploid fibroblasts 2BS, and the promoter activities were assayed. The results showed that the mutants of either GC Ⅰ, GC Ⅱ or GC Ⅳ decreased p16 promoter activity, whereas little effect by mutation of GC Ⅲ and GC Ⅴ was found. Electrophoretic mobility shift assay (EMSA) confirmed that transcription factor Sp1/Sp3 could bind GC Ⅰ, GC Ⅱ and GC Ⅳ and that the retarded bands were competed by Sp1 and Sp3 antibodies. Co transfection of Sp1 expression vector increased the promoter activity while the activity was slightly decreased by Sp3. The results indicated that transcription factors Sp1 and Sp3 were involved in the transcriptional regulation of p16.

关 键 词：人成纤维细胞转录因子Sp1/Sp3 人胚肺二倍体成纤维细胞(2BS) 电泳迁移率变动分析(EMSA) 细胞周期蛋白依赖激酶(cdk) 

分 类 号：Q756[生物学—分子生物学]