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 .     

Cell cycle specific changes in the human cyclin B1 gene regulatory region as revealed by response to trichostatin A

The human cyclin Bl gene is cell cycle regulated with maximal activity during G(2)/M. We examined the role of histone deacetylation in cyclin Bl regulation using the histone deacetylase inhibitor trichostatin A (TSA). TSA treatment (100 ng/ml) of NIH3T3 cells containing the luciferase reporter construct pCycB(-287)-LUC caused an increase in promoter activity in G(0) and G(1) but no significant change in G(2). Removal of upstream sequences including an E-box and Sp1 site eliminated the TSA induced increase in G(0) and G(1), and caused a decrease in promoter activity during S and G(2). Promoter activity increased only 2-fold following TSA treatment of G(0) cells containing the construct pCycB(MUT-E-Box)-LUC with an E-box mutation, and a decrease in activity was detected during G(2). We conclude that histone deacetylation contributes to the repression of cyclin B1 expression in G(0) and G(1), and that this mechanism requires, in part, the E-box. TSA reduction of cyclin B1 promoter activity in G(2), however, involves sequences within the first 119 bp. A working model for cyclin B1 regulation is provided.     

Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) cells

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G(1) to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Béréziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349-26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) -136 and -96 and between bp -29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the -29 to +139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.