人成纤维细胞转录因子Sp1Sp3对p16~(INK4a)基因的调控

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 .     

Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene.

The cDNAs for two DNA binding proteins of BTE, a GC box sequence in the promoter region of the P-450IA1(CYP1A1) gene, have been isolated from a rat liver cDNA library by using the BTE sequence as a binding probe. While one is for the rat equivalent to human Sp1, the other encodes a primary structure of 244 amino acids, a novel DNA binding protein designated BTEB. Both proteins contain a zinc finger domain of Cys-Cys/His-His motif that is repeated three times with sequence similarity of 72% to each other, otherwise they share little or no similarity. The function of BTEB was analysed by transfection of plasmids expressing BTEB and/or Sp1 with appropriate reporter plasmids into a monkey cell line CV-1 and compared with Sp1. BTEB and Sp1 activated the expression of genes with repeated GC box sequences in promoters such as the simian virus 40 early promoter and the human immunodeficiency virus-1 long terminal repeat promoter. In contrast, BTEB repressed the activity of a promoter containing BTE, a single GC box of the CYP1A1 gene that is stimulated by Sp1. When the BTE sequence was repeated five times, however, BTEB turned out to be an activator of the promoter. RNA blot analysis showed that mRNAs for BTEB and Sp1 were expressed in all tissues tested, but their concentrations varied independently in tissues. The former mRNA was rich in the brain, kidney, lung and testis, while the latter was relatively abundant in the thymus and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)