肿瘤抑制蛋白p53具有转录激活活性,被认为可以介导其生物学功能,包括G1阻滞和凋亡。要了解更多关于p53的反式激活因子在体内的功能,我们进行了基因组足迹实验,检查p53调控基因p21,GADD45和MDM2调控区的p53-DNA相互作用.使用电离辐射诱导人ML-1粒细胞白血病细胞的DNA损伤,检查了这些包含p53共有结合位点的基因的启动子和内含子区域的体内足迹。照射后,p53蛋白的表达均匀且持续,p21,GADD45和MDM2mRNA的强烈诱导。在p21启动子中的两个p53共有结合位点,在照射后1至2小时开始的照射细胞中观察到减少的DNaseI裂解,在2小时后最明显,在8小时后减少。在照射后2小时开始,在GADD45基因的第三个内含子中也观察到部分体内足迹。在MDM2基因中的两个p53结合位点处没有看到体内足迹。我们的研究提供了直接证据,表明p53的DNA损伤诱导活性是由其在p21和GADD45基因中的共有DNA结合位点介导的。我们建议,体内p53-DNA相互作用的瞬时性质和相对不稳定性可能使p53蛋白更容易进入快速周转途径,该途径在蛋白质稳定结合DNA的条件下可能会受到损害。
The tumor suppressor protein p53 has a transcriptional activation activity thought to mediate its biologic function including G1 arrest and perhaps apoptosis. To learn more about p53\'s transactivator function in vivo, we performed genomic footprinting experiments examining p53-DNA interactions in the regulatory regions of the p53-regulated genes p21, GADD45, and MDM2. Using ionizing radiation to induce DNA damage in human ML-1 myeloblastic leukemia cells, the promoter and intronic regions of these genes containing p53-
consensus binding sites were examined for in vivo footprints. There was a uniform and sustained expression of p53 protein as well as a strong induction of p21, GADD45, and MDM2 mRNA following irradiation. At the two p53
consensus binding sites in the p21 promoter, reduced DNaseI cleavage was observed in irradiated cells beginning 1 to 2h after irradiation, being most pronounced after 2 h and diminishing after 8 h. A partial in vivo footprint was also observed in the third intron of the GADD45 gene beginning 2 h after irradiation. No in vivo footprints were seen at the two p53 binding sites in the MDM2 gene. Our study provides direct evidence that the DNA damage-induced activity of p53 is mediated by its
consensus DNA binding sites in the p21 and GADD45 genes. We suggest that the transient nature and relative instability of p53-DNA interactions in vivo may make the p53 protein more accessible to a rapid turnover pathway which might be impaired under conditions when the protein is stably bound to DNA.