Xiao G, White D, Bargonetti J
The Institute for Biomolecular Structure and Function, and Department of Biological Sciences, Hunter College, New York, NY 10021, USA.
Oncogene. 1998 Mar 5;16(9):1171-81. doi: 10.1038/sj.onc.1201631.
The mdm2 oncogene is a p53 responsive gene which contains both a p53 independent and a p53 dependent promoter (P1 and P2 respectively). We have utilized ligation mediated PCR genomic footprinting in order to investigate the intra-nuclear binding of p53 to the mdm2 P2 promoter. The DNase I protection pattern in nuclei from murine cells lacking p53 has been compared to the protection pattern in cells containing a temperature sensitive p53-Val135. At 32 degrees C p53-Val135 assumes a wild-type conformation while at 37 degrees C this p53 is conformationally mutant. We observed clear wild-type p53 dependent protection of the putative p53 response elements (REs) as well as protection of the adjacent TATA box. Interestingly the protection pattern observed with purified wild-type p53 on naked DNA showed less nucleotide sequence protection than the protection observed to be p53 dependent in nuclei. Constitutive DNase I hypersensitivity at both the mdm2 P1 and P2 promoters was detected by indirect Southern blot analysis. DNase I hypersensitivity reflects altered chromatin conformations resulting, most likely, from the absence of nucleosomes. Taken together our findings suggest that the mdm2 P2 promoter is maintained in a nucleosome free state which is pre-primed for transcriptional activation by p53.
mdm2癌基因是一种p53反应基因，它包含一个不依赖p53的启动子和一个依赖p53的启动子（分别为P1和P2）。我们利用连接介导的PCR基因组足迹法来研究p53在细胞核内与mdm2 P2启动子的结合情况。将缺乏p53的小鼠细胞核中的DNase I保护模式与含有温度敏感型p53-Val135的细胞中的保护模式进行了比较。在32℃时，p53-Val135呈现野生型构象，而在37℃时，这种p53是构象突变型。我们观察到假定的p53反应元件（REs）有明显的野生型p53依赖性保护，以及相邻TATA框的保护。有趣的是，用纯化的野生型p53对裸DNA进行观察时，所观察到的核苷酸序列保护比在细胞核中观察到的p53依赖性保护要少。通过间接Southern印迹分析检测到mdm2 P1和P2启动子处均存在组成型DNase I超敏性。DNase I超敏性反映了染色质构象的改变，这很可能是由于核小体的缺失所致。综合我们的研究结果表明，mdm2 P2启动子维持在无核小体状态，这为p53的转录激活做好了准备。
