人CGL-1/细胞毒性丝氨酸蛋白酶B基因(CSP-B;也称为颗粒酶B)在细胞毒性T淋巴细胞成熟期间被转录激活。通过用12-O-十四烷酰佛波醇-13-乙酸酯(TPA)和N6-2\'-O-二丁酰腺苷3\'处理,可以在PEERT细胞白血病细胞系中模拟活化,5'-环单磷酸酯(bt2cAMP)。在这份报告中,我们表明,在TPAbt2cAMP刺激的PEER细胞中,CPS-B启动子的转录激活需要位于CSP-B转录起始位点5'的共有AP-1元件和共有cAMP反应元件(CRE)。包含两个元件的94-bp片段以不依赖于方向的方式激活异源启动子。AP-1位点中的几个单核苷酸取代消除了94bp片段的活性。共有CRE中的几个点突变大大降低了启动子活性,但是一个CRE突变会使活性增加四倍。用第二拷贝的AP-1位点替换CRE导致与野生型序列相当的转录活性水平。但用CRE替换AP-1位点会消除活性。AP-1站点和CRE都不能有效地用SP-1站点代替。AP-1位点和CRE之间的缺失仅在保留螺旋间距的情况下保留全部活性,这表明这两个元件之间的协同作用是因子与DNA的协同结合或DNA结合因子与另一种蛋白质的协同结合的结果。
The human CGL-1/cytotoxic serine protease B gene (CSP-B; also known as granzyme B) is transcriptionally activated during cytotoxic T-lymphocyte maturation. Activation can be mimicked in the PEER T-cell leukemia cell line by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) and N6-2\'-O-dibutyryladenosine 3\',5\'-cyclic monophosphate (bt2cAMP). In this report, we show that a
consensus AP-1 element and a
consensus cAMP response element (CRE) located 5\' to the CSP-B transcriptional start site are both required for transcriptional activation of the CPS-B promoter in TPA + bt2cAMP-stimulated PEER cells. A 94-bp fragment containing both elements activates a heterologous promoter in an orientation-independent fashion. Several single nucleotide substitutions in the AP-1 site abolish activity of the 94-bp fragment. Several point mutations in the
consensus CRE substantially reduce promoter activity, but one CRE mutation increases activity fourfold. Replacement of the CRE with a second copy of the AP-1 site results in a level of transcriptional activity comparable with that of the wild-type sequence, but replacement of the AP-1 site with a CRE abolishes activity. Neither the AP-1 site nor the CRE can be effectively replaced with an SP-1 site. Deletions between the AP-1 site and the CRE retain full activity only if helical spacing is preserved, suggesting that synergism between these two elements is either the result of cooperative binding of factors to the DNA or of cooperative binding of DNA-bound factors to another protein.